EP2470559A1 - Compositions de facteur ix de coagulation et leurs procédés de fabrication et d'utilisation - Google Patents

Compositions de facteur ix de coagulation et leurs procédés de fabrication et d'utilisation

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Publication number
EP2470559A1
EP2470559A1 EP10814049A EP10814049A EP2470559A1 EP 2470559 A1 EP2470559 A1 EP 2470559A1 EP 10814049 A EP10814049 A EP 10814049A EP 10814049 A EP10814049 A EP 10814049A EP 2470559 A1 EP2470559 A1 EP 2470559A1
Authority
EP
European Patent Office
Prior art keywords
xten
factor
sequence
polypeptide
cfxten
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP10814049A
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German (de)
English (en)
Other versions
EP2470559B1 (fr
EP2470559A4 (fr
Inventor
Volker Schellenberger
Joshua Silverman
Willem Peter Stemmer
Chia-Wei Wang
Benjamin Spink
Nathan Carl Geething
Wayne To
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Amunix Pharmaceuticals Inc
Original Assignee
Amunix Operating Inc
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Publication date
Priority claimed from US12/699,761 external-priority patent/US8673860B2/en
Application filed by Amunix Operating Inc filed Critical Amunix Operating Inc
Priority to EP17155615.2A priority Critical patent/EP3222287A1/fr
Publication of EP2470559A1 publication Critical patent/EP2470559A1/fr
Publication of EP2470559A4 publication Critical patent/EP2470559A4/fr
Application granted granted Critical
Publication of EP2470559B1 publication Critical patent/EP2470559B1/fr
Not-in-force legal-status Critical Current
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/644—Coagulation factor IXa (3.4.21.22)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6437—Coagulation factor VIIa (3.4.21.21)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21022—Coagulation factor IXa (3.4.21.22)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli

Definitions

  • FIX Human factor DC
  • FIX Human factor DC
  • a deficiency of functional FIX due to an X-linked- disorder that occurs in about one in 30,000 males, results in hemophilia B, also known as Christmas disease, named after a young boy named Stephen Christmas who was found to be lacking this factor.
  • FIX hepatitis C virus
  • Factor VII is a coagulation factor protein synthesized in the liver and secreted into the blood as a single chain zymogen with a molecular weight of approximately 50 kDa.
  • the FVII zymogen is converted SGR Dkt. Ref. 32808-726601 into an activated form (FVIIa) by proteolytic cleavage, and the activated form, when complexed with tissue factor (TF), is able to convert both factor IX and factor X into their activated forms, leading to rapid thrombin generation and fibrin formation.
  • Chemical modifications to a therapeutic protein can reduce its in vivo clearance rate and subsequent increase serum half-life.
  • One example of a common modification is the addition of a polyethylene glycol (PEG) moiety, typically coupled to the protein via an aldehyde or N- hydroxysuccinimide (NHS) group on the PEG reacting with an amine group (e.g. lysine side chain or the N-terminus).
  • PEG polyethylene glycol
  • NHS N- hydroxysuccinimide
  • the conjugation step can result in the formation of heterogeneous product mixtures that need to be separated, leading to significant product loss and complexity of manufacturing and does not result in a completely chemically-uniform product.
  • the pharmacologic function of the therapetuics protein may be hampered if amino acid side chains in the vicinity of its binding site are modified by the PEGylation process.
  • Fusing an Fc domain to the therapeutic protein is another approach to increases the size of the therapeutic protein, hence reducing the rate of clearance through the kidney.
  • the Fc domain confers the ability to bind to, and be recycled from lysosomes by, the FcRn receptor, which results in increased pharmacokinetic half-life.
  • the Fc domain does not fold efficiently during recombinant expression, and tends to form insoluble precipitates known as inclusion bodies. These inclusion bodies must be solubilized and functional protein must be renatured from the misfolded aggregate. Such process is time-consuming, inefficient, and expensive. Accordingly, there remains a need for improved coagulation factor compositions with increased half-life which can be administered less frequently, and/or be produced by a simpler process at a cheaper cost.
  • the present invention is directed to compositions and methods for the treatment or improvement of a condition or the enhancement of a parameter associated with the administration of coagulations factors ⁇ and/or VII.
  • the present invention provides compositions of fusion proteins comprising one or more extended recombinant polypeptides (XTEN).
  • XTEN extended recombinant polypeptides
  • a subject XTEN is typically a non-repetitive sequence and unstructured conformation.
  • XTEN is linked to a coagulation factor ("CF") selected from factor EX (“FIX”), factor VII (“FVII”), factor Vll-factor EX hybrids, and sequence variants thereof, resulting in a coagulation factor-XTEN fusion protein ("CFXTEN").
  • CFXTEN coagulation factor
  • the present disclosure is directed to pharmaceutical compositions comprising the fusion proteins and the uses thereof for treating coagulation factor-related diseases, disorders or conditions.
  • the CFXTEN compositions have enhanced pharmacokinetic properties compared to CF not linked to XTEN, which may permit more convenient dosing and improved efficacy.
  • the CFXTEN compositions of the invention do not have a component selected the group consisting of: polyethylene glycol (PEG), albumin, antibody, and an antibody fragment.
  • PEG polyethylene glycol
  • albumin albumin
  • antibody fragment an antibody fragment
  • the invention provides an isolated factor EX fusion protein, comprising a factor ⁇ sequence that is at least about 90%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% identical to an amino acid sequence selected from Table 1.
  • the factor ⁇ having such sequence idendity is further linked to an extended recombinant polypeptide (XTEN) having at least about 100 to about 3000 amino acid residues.
  • XTEN extended recombinant polypeptide
  • the XTEN is linked to the C-terminus of the FIX or the FVII CF.
  • the invention provides an isolated factor VII fusion protein, comprising a factor VII that is at least about 90%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% identical to an amino acid sequence selected from Table 2.
  • the factor VII having such sequence is linked to an extended recombinant polypeptide (XTEN).
  • Non-limiting examples of CFXTEN with a single FIX or a single FVII linked to a single XTEN are presented in Table 41.
  • the invention provides a CFXTEN composition has at least about 80% sequence identity compared to a CFXTEN from Table 41, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% sequence identity as compared to a CFXTEN from Table 41.
  • the CF and the XTEN components of the fusion protein are linked via a cleavage sequence that is cleavable by a protease, including endogenous mammalian proteases.
  • protease include, but are not limited to, FXIa, FXIIa, kallikrein, FVIIa, FD a, FXa, thrombin, elastase-2, granzyme B, MMP-12, MMP-13, MMP-17 or MMP-20, TEV, enterokinase, rhinovirus 3C protease, and sortase A, or a sequence selected from Table 7.
  • a CFXTEN composition with a cleavage sequence has a sequence having at least about 80% sequence identity compared to a CFXTEN from Table 42, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% sequence identity as compared to a CFXTEN from Table 42.
  • the invention also provides substitution of any of the CF sequences of Table 1 or Table 2 for a CF in a sequence of Table 42, and substitution of any XTEN sequence of Table 4 for an XTEN in a sequence of Table 42, and substitution of any cleavage sequence of Table 7 for a cleavage sequence in a sequence of Table 42.
  • CFXTEN embodiments having cleavage sequences cleavage of the cleavage sequence by the protease releases the XTEN from the CF.
  • the CF component becomes biologically active or has an increase in activity upon its release from the XTEN by cleavage of the cleavage sequence, wherein the pro-coagulant activity is at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% compared to the corresponding FIX or FVII not linked to XTEN.
  • the invention provides isolated CFXTEN fusion proteins that comprise a second XTEN of about 36 to about 3000 amino acid residues, which can be identical or can be different from the first XTEN, wherein the second XTEN can be incorporated between any two adjacent domains of the CF, i.e., between the Gla, EFG1, EGF2, activating peptide and protease domains, or is incorporated within the sequence of an existing loop domain of a domain sequence of the CF, as described more fully in the Examples.
  • the first and the second XTEN can be an amino acid sequence selected WSGR Dkt. Ref.
  • the isolated fusion protein comprises a second XTEN of about 36 to about 3000 amino acid residues.
  • the fusion protein can adopt a multiple-XTEN configuration of Table 6, or a variation thereof.
  • the invention provides CFXTEN compositions comprising XTEN linked to a factor VII comprising one or more heterologous cleavage sequences cleavable by the same or different pro- coagulant proteases.
  • the factor VII comprises a heterologous sequence of factor XI incorporated into or substituted for portions of the FVII sequence, resulting in factor VTi-factor DC hybrid sequence variants.
  • a portion or the entirety of the sequence from the activation peptide domain of FIX is incorporated or substituted for FVII sequences bridging the region between the EFG2 and protease domains of the FVII component, resulting in compositions that can be activated as part of the intrinsic system of the coagulation cascade (e.g., activated factor XI).
  • the factor Vll-factor IX CFXTEN composition can be activated by a pro-coagulant protease in the absence of tissue factor, such that the CFXTEN can serve as a by-pass of factors VIH and DC in the intrinsic coagulation pathway when such factors are deficient (e.g., in hemophilia A or B) or when inhibitors to these factors are present.
  • the FVII-FDC sequence variant incorporates the full-length FDC AP domain plus at least about 2, or at least about 3, or at least about 4, or at least about 5, or at least about 6, or at least about 7, or at least about 8, or at least about 9, or at least about 10, or at least about 11, or at least about 12 or moreamino acids flanking adjacent amino acid residues on one or both sides of the R145-A146 and R180-V181 cleavage sites of the FDC AP domain (e.g., the sequence
  • the CFXTEN FVII-FDC sequence variant comprises a heterologous FDC sequence exhibiting at least at least about 80%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or 100% identity compared to the sequence:
  • the CFXTEN comprises FVII-FDC sequence variants that incorporate a portion of the FDC AP that includes a sequence of at least about 2, or at least about 3, or at least about 4, or at least about 5, or more amino acids that flank one or both sides of the R145-A146 cleavage site (e.g., the sequence TSKLTRAETVFP in the case of 6 flanking amino acids on either side of the cleavage site) or a sequence of at least about 2, or at least about 3, or at least about 4, or at least about 5 or more amino acids that flank one or both sides of the R180-V181 cleavage site (e.g., the sequence and DFTRV in the case of 4 amino acids on the N-terminal flank and valine as the C-terminus of the cleavage site from WSGR Dkt. Ref. 32808-726601
  • the CFXTEN FVII-FIX sequence variant comprises a heterologous FIX sequence exhibiting at least at least about 80%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or 100% identity compared to a sequence selected from TSKLTRAETVFP and FNDFTRV, when optimally aligned.
  • the CFXTEN comprises a FVII-FIX sequence variant disclosed above that further includes the same AP cleavage sequence as a linker between the C-terminus of the FVII component and the XTEN component of the fusion protein, e.g., an N- to C-terminus configuration of FVII variant-AP sequence-XTEN, thereby permitting the release of the FVII variant component from the CFXTEN fusion protein when cleaved by the same pro-coagulant protease as per that of the FVII to FVIIa transition.
  • the FVII-FIX CFXTEN of any of the foregoing embodiments includes the factor XI cleavage sequence KLTRAET as the linker between the FVII-FIX sequence and the XTEN, thereby permitting the release of the FVII variant component from the CFXTEN fusion protein by the initiation of the intrinsic coagulation cascade.
  • the invention provides a CFXTEN with a FVII-FIX hybrid sequence that exhibits at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, sequence identity compared to a sequence from Table 43.
  • the invention provides a FVII-FIX sequence variant with incorporated FIX-derived AP cleavage sequence that is not linked to an XTEN.
  • the FVII-FIX sequence without an XTEN exhibits at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity as compared with a sequence from Table 43 without an XTEN.
  • the invention provides a fusion protein of formula I:
  • CF is a coagulation factor
  • x is either 0 or 1
  • y is either 0 or 1 wherein x+y >1
  • XTEN is an extended recombinant polypeptide
  • the invention provides a fusion protein of formula II:
  • CF is a coagulation factor a
  • S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence
  • x is either 0 or 1 and y is either 0 or 1 wherein x+y >1
  • XTEN is an extended recombinant polypeptide.
  • the invention provides an isolated fusion protein, wherein the fusion protein is of formula III:
  • XTEN x -(S) x -(CF)-(S) y -(XTEN) y ⁇ WSGR Dkt.
  • CF a coagulation factor
  • S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence
  • x is either 0 or 1
  • y is either 0 or 1 wherein x+y >1
  • XTEN is an extended recombinant polypeptide.
  • the invention provides an isolated fusion protein of formula IV:
  • Gla is a Gla domain of FIX
  • EGF 1 is an EGF1 domain of FIX
  • EGF2 is an EFG2 domain of FIX
  • AP is an activator peptide of FDi
  • PRO is a protease domain of FIX
  • S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence
  • u is either 0 or 1
  • v is either 0 or 1
  • x is either 0 or 1
  • y is either 0 or 1
  • z is either 0 or lwith the proviso that u + v + w + x + z >l
  • XTEN is an extended recombinant polypeptide.
  • the invention provides an isolated fusion protein of formula V:
  • Gla is a Gla domain of FIX
  • EGF1 is an EGF1 domain of FIX
  • EGF2 is an EFG2 domain of FIX
  • API is the N-terminal sequence portion of the activator peptide domain of FIX that includes a first native cleavage sequence of the AP domain
  • AP2 is the C-terminal sequence portion of the activator peptide domain of FIX that includes a second native cleavage sequence of the AP domain
  • PRO is a protease domain of FIX
  • S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence
  • u is either 0 or 1
  • v is either 0 or 1
  • x is either 0 or 1
  • y is either 0 or 1
  • z is either 0 or 1 with the proviso that u + v + w + x + z > 1
  • XTEN is an extended recombinant polypeptid
  • the invention provides an isolated fusion protein of formula VI:
  • Gla is a Gla domain of FVII
  • EGF 1 is an EGF 1 domain of FVII
  • EGF2 is an EFG2 domain of FVII
  • PRO is a protease domain of FVII
  • S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence
  • u is either 0 or 1
  • v is either 0 or 1
  • x is either 0 or 1
  • y is either 0 or 1 with the proviso that u + v + w + y >l
  • XTEN is an extended recombinant polypeptide.
  • the invention provides an isolated fusion protein of formula VII:
  • Gla is a Gla domain of FVII
  • EGF 1 is an EGF 1 domain of FVII
  • EGF2 is an EFG2 domain of FVII
  • PRO is a protease domain of FVII
  • API is the N-terminal WSGR Dkt. Ref.
  • XTEN is an extended recombinant polypeptide.
  • the CFXTEN composition can include the entirety of the FIX activator peptide domain sequence or one or both cleavage sequences from the activator peptide domain of factor ⁇ , e.g., a sequence of at least about 3 to about 12 amino acids that flank the R145-A146 cleavage site and the sequence of at least about 1 to about 5 amino acids that flank the R180-V 181 cleavage site, as described more fully above.
  • the invention also contemplates substitution of any of the other cleavage sequences of Table 7 for the AP cleavage sequences.
  • the CFXTEN compositions of the embodiments described herein can be evaluated for retention of activity (including after cleavage of any incorporated XTEN-releasing cleavage sites) using any appropriate in vitro assay disclosed herein (e.g., the assays of Table 40 or the assays described in the Examples), to determine the suitability of the configuration for use as a therapeutic agent in the treatment of a coagulation-factor related disease, disorder or condition.
  • the CFXTEN exhibits at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% of the activity compared to the native CF not linked to XTEN.
  • the CF component released from the CFXTEN by enzymatic cleavage of the incorporated cleavage sequence linking the CF and XTEN components exhibits at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% of the activity compared to the native CF not linked to XTEN.
  • the XTEN of the CFXTEN compositions have at least about 200, or at least about 400, or at least about 800, or at least about 900, or at least about 1000, or at least about 2000, up to about 3000 amino acids residues.
  • the XTEN of the CFXTEN fusion protein compositions is characterized in that they have one or more of the following characteristics: (a) at least a first XTEN comprises at least about 200 contiguous amino acids that exhibits at least about 90%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% identity to a comparable length of an amino acid sequence selected from a sequence shown in Table 4; (b) the XTEN sequence lacks a predicted T-cell epitope when analyzed by TEPITOPE algorithm, wherein the TEPITOPE algorithm prediction for epitopes within the XTEN sequence is based on a score of -5, or -6, or -7, or -8, or -9 or greater; (c) the XTEN has
  • Ref. 32808-726601 glutamate (E) and proline (P) residues constitutes more than about 90%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% of the total amino acid residues of the XTEN.
  • the invention provides CFXTEN fusion proteins, wherein the XTEN is characterized in that the sum of asparagine and glutamine residues is less than 10% of the total amino acid sequence of the XTEN, the sum of methionine and tryptophan residues is less than 2% of the total amino acid sequence of the XTEN, the XTEN sequence has less than 5% amino acid residues with a positive charge, the XTEN sequence has greater than 90% random coil formation, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% random coil formation as determined by GOR algorithm; and the XTEN sequence has less than 2% alpha helices and 2% beta-sheets as determined by the Chou-Fasman algorithm. In some embodiments, no one type of amino acid constitutes more than 30% of the XTEN sequence of the CFXTEN.
  • the invention provides CFXTEN fusion proteins, wherein the XTEN is characterized in that at least about 80%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% of the XTEN sequence consists of non- overlapping sequence motifs wherein each of the sequence motifs has about 9 to about 14 amino acid residues and wherein the sequence of any two contiguous amino acid residues does not occur more than twice in each of the sequence motifs consist of four to six types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P).
  • G glycine
  • A alanine
  • S serine
  • T threonine
  • E glutamate
  • P proline
  • the XTEN is characterized in that at least about 80%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% of the XTEN sequence consists of non- overlapping sequence motifs wherein the motifs are selected from Table 3.
  • the XTEN has a sequence in which no three contiguous amino acids are identical unless the amino acid is serine, in which case no more than three contiguous amino acids are serine residues.
  • the XTEN component of the CFXTEN has a subsequence score of less than 10, or less than 9, or less than 8, or less than 7, or less than 6, or less than 5, or less. In the embodiments of this paragraph, the XTEN is characterized as "substantially non-repetitive.”
  • the invention provides CFXTEN comprising at least a second XTEN, wherein the XTEN sequence exhibits at least about 80%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity compared to a sequence from Table 4, Table 9, Table 10, Table 11, Table 12, or Table 13.
  • CFXTEN fusion proteins exhibits enhanced pharmacokinetic properties compared to CF not linked to XTEN, wherein the enhanced properties include but are not limited to longer terminal half-life, larger area under the curve, increased time in which the blood concentration remains within the therapeutic window, increased time between consecutive doses results in blood WSGR Dkt.
  • the enhanced properties include but are not limited to longer terminal half-life, larger area under the curve, increased time in which the blood concentration remains within the therapeutic window, increased time between consecutive doses results in blood WSGR Dkt.
  • Ref. 32808-726601 concentrations within the therapeutic window and decreased dose in moles over time that can be administered compared to a CF not linked to XTEN, yet still result in a blood concentration within the therapeutic window for that composition.
  • the terminal half-life of the CFXTEN fusion protein administered to a subject is increased at least about three-fold, or at least about four-fold, or at least about five-fold, or at least about six-fold, or at least about eight-fold, or at least about ten-fold, or at least about 20-fold, or at least about 40-fold, or at least about 60-fold, or at least about 100-fold, or even higher as compared to CF not linked to XTEN and administered to a subject at a comparable dose.
  • the terminal half-life of the CFXTEN fusion protein administered to a subject is at least about 12 h, or at least about 24 h, or at least about 48 h, or at least about 72 h, or at least about 96 h, or at least about 120 h, or at least about 144 h, or at least about 21 days or greater.
  • the enhanced pharmacokinetic property is reflected by the fact that the blood concentrations that remain within the therapeutic window for the CFXTEN fusion protein for a given period are at least about two fold, or at least about three-fold, or at least about four-fold, or at least about five-fold, or at least about six-fold, or at least about eight-fold, or at least about ten-fold longer, or at least about 20-fold, or at least about 40-fold, or at least about 60-fold, or at least about 100-fold compared to CF not linked to XTEN and administered to a subject at a comparable dose.
  • administration of a CFXTEN to a subject using a therapeutically-effective dose regimen results in a gain in time of at least two-fold, or at least three-fold, or at least four-fold, or at least five-fold, or at least six-fold, or at least eight-fold, or at least 10-fold, or at least about 20-fold, or at least about 40- fold, or at least about 60- fold, or at least about 100-fold or higher between at least two consecutive C max peaks and or troughs for blood levels of the fusion protein compared to the corresponding CF not linked to the XTEN and administered using a comparable dose regimen to a subject.
  • the XTEN enhances thermostability of CF when linked to the XTEN wherein the thermostability is ascertained by measuring the retention of biological activity after exposure to a temperature of about 37°C for at least about 7 days of the biologically active protein in comparison to the biologically active protein not linked to the XTEN.
  • the retention of biological activity increases by at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, or about 150%, at least about 200%, at least about 300%, or about 500% longer compared to the CF not linked to the XTEN.
  • the isolated CFXTEN fusion protein is configures to have reduced binding affinity for a clearance receptor as compared to the corresponding CF not linked to the XTEN.
  • the CFXTEN fusion protein exhibits binding affinity for a clearance receptor of the CF in the range of about 0.01%-30%, or about 0.1% to about 20%, or about 1% to about 15%, or about 2% to about 10% of the binding affinity of the corresponding CF not linked to the XTEN.
  • a CFXTEN fusion protein with reduced affinity can have reduced active clearance and a WSGR Dkt. Ref.
  • 32808-726601 corresponding increase in half-life of at least about 3-fold, or at least about 5-fold, or at least about 6-fold, or at least about 7-fold, or at least about 8-fold,or at least about 9-fold, or at least about 10-fold, or at least about 12-fold, or at least about 15-fold, or at least about 17-fold, or at least about 20-fold, or at least about 30-fold, or at least about 50-fold, or at least about 100-fold longer compared to the corresponding CF that is not linked to the XTEN.
  • the invention provides CFXTEN fusion proteins wherein the CFXTEN exhibits increased solubility of at least three-fold, or at least about four-fold, or at least about five-fold, or at least about six-fold, or at least about seven-fold, or at least about eight-fold, or at least about nine-fold, or at least about ten-fold, or at least about 15-fold, or at least a 20-fold, or at least 40-fold, or at least 60- fold at physiologic conditions compared to the CF not linked to XTEN.
  • CFXTEN fusion proteins exhibit an increased apparent molecular weight as determined by size exclusion chromatography, compared to the actual molecular weight.
  • the CF comprising a FIX and at least a first XTEN exhibits an apparent molecular weight of at least about 400 kD, or at least about 500 kD, or at least about 700 kD, or at least about 1000 kD, or at least about 1400 kD, or at least about 1600 kD, or at least about 1800kD, or at least about 2000 kD, while the actual molecular weight of each FD component of the fusion protein is about 50 kD and the molecular weight of the fusion protein ranges from about 70 to about 125 kDa.
  • the CF comprising a FVII and at least a first XTEN exhibits an apparent molecular weight of at least about 400 kD, or at least about 500 kD, or at least about 700 kD, or at least about 1000 kD, or at least about 1400 kD, or at least about 1600 kD, or at least about 1800kD, or at least about 2000 kD, while the actual molecular weight of each FIX component of the fusion protein is about 50 kD and the molecular weight of the fusion protein ranges from about 70 to about 125 kDa.
  • the CFXTEN fusion proteins can have an apparent molecular weight that is about 6-fold greater, or about 8-fold greater, or about 10-fold greater, or about 12-fold greater, or about 15-fold greater than the actual molecular weight of the fusion protein.
  • the isolated CFXTEN fusion protein of any of the embodiments disclosed herein exhibit an apparent molecular weight factor under physiologic conditions that is greater than about 4, or about 5, or about 6, or about 7, or about 8, or about 10, or greater than about 15.
  • administration of a therapeutically effective dose of a fusion protein of one of formulae I-VII to a subject in need thereof can result in a gain in time of at least two-fold, or at least three-fold, or at least four-fold, or at least five-fold or more spent within a therapeutic window for the fusion protein compared to the corresponding CF not linked to the XTEN of and administered at a comparable dose to a subject.
  • administration of a therapeutically effective dose of a fusion protein of an embodiment of formulas I-VII to a subject in need thereof can result in a gain in time between consecutive doses necessary to maintain a therapeutically effective dose regimen of at least 48 h, or at least 72 h, or at least about 96 h, or at least about 120 h, or at least about 7 days, or at least about 14 days, or at least about 21 days between consecutive doses compared to a CF not linked to XTEN and administered at a comparable dose.
  • fusion proteins of the disclosed compositions can be designed to have different
  • configurations, N- to C-terminus, of a CF and XTEN and optional spacer sequences including but not limited to XTEN-CF, CF-XTEN, XTEN-S-CF, CF-S-XTEN, XTEN-CF-XTEN, CF-CF-XTEN, XTEN- CF-CF, CF-S-CF-XTEN, XTEN-CF-S-CF, and multimers thereof.
  • the choice of configuration can, as disclosed herein, confer particular pharmacokinetic, physico/chemical, or pharmacologic properties including, in the case of an incorporated cleavage sequence, the release of the CF with a concomitant increase in activity.
  • the CFXTEN fusion protein is characterized in that: (i) it has a longer half-life when administered to a subject compared to the corresponding coagulation factor not linked to the XTEN administered to a subject under an otherwise equivalent dose; (ii) when a smaller molar amount of the fusion protein is administered to a subject in comparison to the corresponding coagulation factor that lacks the XTEN administered to a subject under an otherwise equivalent dose regimen, the fusion protein achieves a comparable area under the curve (AUC) as the corresponding coagulation factor not linked to the XTEN; (iii) when a smaller molar amount of the fusion protein is administered to a subject in comparison to the corresponding coagulation factor that lacks the XTEN administered to a subject under an otherwise equivalent dose regimen, the fusion protein achieves a comparable therapeutic effect as the corresponding coagulation factor not linked to the XTEN; (iv) when the fusion protein is administered to a subject less frequently in comparison to the a
  • the fusion protein achieves comparable area under the curve (AUC) as the corresponding coagulation factor not linked to the XTEN; or (vii) when an accumulatively smaller molar amount of the fusion protein is administered to a subject in comparison to the corresponding coagulation factor not linked to the XTEN administered to a subject under an otherwise equivalent dose period, the fusion protein achieves comparable therapeutic effect as the corresponding coagulation factor not linked to the XTEN.
  • the invention provides a method of producing a fusion protein comprising a factor VII or factor IX or a factor VTI-factor ⁇ hybrid coagulation factor fused to one or more extended recombinant polypeptides (XTEN), comprising: (a) providing host cell comprising a recombinant polynucleotide molecule encoding the fusion protein (b) culturing the host cell under conditions permitting the expression of the fusion protein; and (c) recovering the fusion protein from the culture.
  • the coagulation factor of the fusion protein has at least 90% sequence identity WSGR Dkt. Ref. 32808-726601 compared to a sequence selected from Table 1 or Table 2.
  • the one or more XTEN of the expressed fusion protein has at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% to about 100% sequence identity compared to a sequence selected from Table 4.
  • the host cell is a eukaryotic cell.
  • the host cell is CHO cell.
  • the isolated fusion protein is recovered from the host cell cytoplasm in substantially soluble form.
  • the invention provides isolated nucleic acids comprising a polynucleotide sequence selected from (a) a polynucleotide encoding the fusion protein of any of the foregoing embodiments, or (b) the complement of the polynucleotide of (a).
  • the invention provides an isolated nucleic acid comprising a polynucleotide sequence that has at least 80% sequence identity, or about 85%, or at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% to about 100% sequence identity compared to (a) a polynucleotide sequence of comparable length selected from Table 41 and Table 42; or (b) the complement of the polynucleotide of (a).
  • the invention provides expression vectors comprising the nucleic acid of any of the embodiments hereinabove described in this paragraph.
  • the expression vector of the foregoing further comprises a recombinant regulatory sequence operably linked to the polynucleotide sequence.
  • the polynucleotide sequence of the expression vectors of the foregoing is fused in frame to a polynucleotide encoding a secretion signal sequence, which can be a CF native signal sequence.
  • the invention provides a host cell that comprises an expression vector of any of the embodiments hereinabove described in this paragraph.
  • the host cell is a eukaryotic cell.
  • the host cell is a CHO cell.
  • the host cell is HE cell.
  • the invention provides pharmaceutical compositions comprising the fusion protein of any of the foregoing embodiments and a pharmaceutically acceptable carrier.
  • the invention provides kits, comprising packaging material and at least a first container comprising the pharmaceutical composition of the foregoing embodiment and a label identifying the pharmaceutical composition and storage and handling conditions, and a sheet of instructions for the reconstitution and/or administration of the pharmaceutical compositions to a subject.
  • the invention provides a method of treating a coagulopathy or a coagulation factor-related disease, disorder or condition in a subject, comprising administering to the subject a therapeutically effective amount of a CFXTEN fusion protein of any of the foregoing embodiments.
  • the coagulation-factor related condition is selected from bleeding disorders (e.g., defective platelet function, thrombocytopenia or von Willebrand's disease), coagulopathies (any disorder of blood coagulation, including coagulation factor deficiencies), hemophilia B (aka Christmas disease), factor DC- related bleeding disorders, factor VII deficiency, hemophilia A, vascular injury, uncontrolled bleeding in subjects not suffering from hemophilia, bleeding from trauma or surgery, bleeding due to anticoagulant WSGR Dkt. Ref. 32808-726601 therapy, and bleeding due to liver disease.
  • the coagulopathy is hemophilia A.
  • the coagulopathy is hemophilia B.
  • the coagulopathy is factor VII deficiency.
  • the CFXTEN is administered to a subject to control a bleeding episode.
  • a CFXTEN comprising a factor Vll-factor IX sequence hybrid is administered to a subject to control a bleeding episode, wherein the CFXTEN is activated by a pro-coagulant protease of the intrinsic coaguation cascade (e.g., activated factor XI).
  • the present invention provides a method of treating a clotting factor deficiency in a subjkect, comprising: administering to said subject a composition comprising a therapeutically effective amount of the factor VII provided herein.
  • the composition can be administered subcutaneously, intramuscularly, or intravenously.
  • the composition is administered at a therapeutically effective amount, weherein the administration results in a gain in time spent within a therapeutic window for the fusion protein compared to the corresponding CF of the fusion protein not linked to the XTEN and administered at a comparable dose to a subject.
  • the gain in time spent within the therapeutic window can at least three-fold longer than the corresponding CF not linked to the XTEN, or alternatively, at least four-fold, or five-fold, or six-fold, or seven-fold, or eight-fold, or nine-fold, or at least 10-fold, or at least 20-fold, or at least about 30-fold, or at least about 50-fold, or at least about 100-fold longer than the corresponding CF not linked to XTEN.
  • a smaller molar amount of e.g.
  • the fusion protein is administered in comparison to the corresponding coagulation factor not linked to the XTEN under an otherwise same dose regimen, and the fusion protein achieves a comparable area under the curve and/or a comparable therapeutic effect as the corresponding coagulation factor not linked to the XTEN; (ii) the fusion protein is administered less frequently (e.g., every two days, about every seven days, about every 14 days, about every 21 days, or about, monthly) in comparison to the corresponding coagulation factor not linked to the XTEN under an otherwise same dose amount, and the fusion protein achieves a comparable area under the curve and/or a comparable therapeutic effect as the corresponding coagulation factor not linked to the XTEN; or (iii) an accumulative smaller molar amount (e.g.
  • the fusion protein achieves a comparable area under the curve and/or a comparable therapeutic effect as the corresponding coagulation factor not linked to the XTEN.
  • the accumulative smaller molar amount is measured for a period of at least about one week, or about 14 days, or about 21 days, or about one month.
  • the therapeutic effect is a measured parameter selected from blood concentrations of WSGR Dkt. Ref. 32808-726601 coagulation factor, prothrombin (PT) assay, activated partial prothrombin (aPTT) assay, bleeding time assay, whole blood clotting time (WBCT), and thrombelastography.
  • invention provides a method of treating a disease, disorder or condition, comprising administering the pharmaceutical composition described above to a subject using multiple consecutive doses of the pharmaceutical composition administered using a therapeutically effective dose regimen.
  • the therapeutically effective dose regimen can result in a gain in time of at least three-fold, or alternatively, at least four-fold, or five-fold, or six-fold, or sevenfold, or eight-fold, or nine-fold, or at least 10-fold, or at least 20-fold, or at least about 30-fold, or at least about 50-fold, or at least about 100-fold longer time between at least two consecutive peaks and/or Cnu- troughs for blood levels of the fusion protein compared to the corresponding CF of the fusion protein not linked to the fusion protein and administered at a comparable dose regimen to a subject.
  • the administration of the fusion protein results in improvement in at least one measured parameter of a coagulation factor-related disease using less frequent dosing or a lower total dosage in moles of the fusion protein of the pharmaceutical composition compared to the corresponding biologically active protein component(s) not linked to the fusion protein and administered to a subject d using a therapeutically effective regimen to a subject.
  • the invention further provides use of the compositions comprising the fusion protein of any of the foregoing embodiments in the preparation of a medicament for treating a disease, disorder or condition in a subject in need thereof.
  • the disease, disorder or condition is selected from group consisting of bleeding disorders, coagulopathies, hemophilia B (aka Christmas disease), factor IX-related bleeding disorders, factor VII deficiency, vascular injury, bleeding from trauma or surgery, bleeding due to anticoagulant therapy, and liver disease. Any of the disclosed embodiments can be practiced alone or in combination depending on the interested application.
  • FIG. 1 shows a schematic representation of exemplary CFXTEN (FDC-XTEN) fusion proteins.
  • FIG. 1 A shows the domain architecture of native FD , with the gamma-carboxyglutamate domain, the EGF1 and EGF2 domains, the activation peptide, and the protease domain, with a linked XTEN at the C- terminus. Arrows indicate the cleavage sites for the activation peptide domain.
  • FIG. IB shows a FIX molecule with an XTEN polypeptide attached to the C-terminus via a cleavage sequence, and indicates a WSGR Dkt Ref. 32808-726601 site for proteolytic cleavage to release the XTEN (arrows indicate the cleavage sites for the activation peptide domain and the release point for the XTEN).
  • FIG. 2 illustrates several examples of CXTEN configurations of FIX-XTEN and associated protease cleavage sites.
  • FIG. 2A shows an FIX-XTEN with two proteolytic cleavage sites (arrows) within the activation peptide of FIX, and a C-terminus XTEN without a cleavage site linkage.
  • FIG. 2B is similar to the configuration of FIG. 2A, but the C-terminus XTEN is linked via a cleavage sequence, with the arrow indicating the release point.
  • FIG. 2C shows three configurations of FIX-XTEN, with the XTEN integrated between the various domains of FIX.
  • FIG. 2A shows an FIX-XTEN with two proteolytic cleavage sites (arrows) within the activation peptide of FIX, and a C-terminus XTEN without a cleavage site linkage.
  • FIG. 2B is similar to the configuration of FIG. 2A, but the
  • FIG. 2D shows an FIX-XTEN with the XTEN portion inserted into the activation peptide between the native cleavage sites, which would release the XTEN upon the proteolytic activation of FIX.
  • FIG. 2E illustrates FIX-XTEN that contain multiple XTEN sequences inserted between different domains with the addition of a releasable XTEN at the C-terminus.
  • FIG. 2F illustrates FIX-XTEN where the XTEN has been inserted within loop domains of FIX.
  • FIG. 3 is a schematic of the coagulation cascade, showing both the extrinsic and intrinsic pathways.
  • FIG. 4 shows several examples of CXTEN configurations of FVII-XTEN.
  • FIG. 4A shows a FVII-XTEN that has not been activated.
  • FIG. 4B shows a FVII-XTEN in which the peptide has been cleaved, resulting in an activated FVIIa-XTEN;
  • FIG. 4C illustrates a FVII-XTEN composition with a cleavage sequence for releasable XTEN in which the FVII component has not been activated, containing a cleavage site for the activation protease (AP) and a second cleavage site for the release protease (RP).
  • FIG. 4D shows a composition of activated FVIIa-XTEN containing a cleavage site for the release protease.
  • FIG. 5 illustrates a strategy for FVII-XTEN design approach using internal XTEN.
  • FIGS. 5A-D show exemplary sites for XTEN insertion between boundaries of the FVII domains with inactive FVII on the left and an activated form of FVII on the right (A: Insertion of XTEN between Gla and EGFl domain, B: Insertion of XTEN between EGFl and EGF2. C: Insertion of XTEN at C-terminus of activation peptide, D: Insertion of XTEN at N-terminus of activation peptide).
  • FIG. 5 illustrates a strategy for FVII-XTEN design approach using internal XTEN.
  • FIGS. 5A-D show exemplary sites for XTEN insertion between boundaries of the FVII domains with inactive FVII on the left and an activated form of FVII on the right (A: Insertion of XTEN between Gla and EGFl domain, B: Insertion of XTEN between EGFl
  • FIG. 5E shows examples of FVII- XTEN in which the XTEN is located within external loops within individual domains fusion proteins, with inactive FVII on the left and FVIIa on the right.
  • the activation peptide in FVII is shown as a thin line versus XTEN that is shown as a fat line.
  • FIG. 6 illustrates essentially the same constructs as FIG. 5, but with an XTEN linked at the C- terminus of each construct.
  • FIG. 7 is a schematic that shows some of the various locations in which XTEN can be inserted internal to the sequences of the coagulation factors FVII or FIX.
  • FIG. 8 is a schematic of the key components of the clotting system.
  • FIG. 7A Normal clotting system with the intrinsic and extrinsic cascade components.
  • FIG. 7 B illustrates a variation in which an inactive/low active form of FVII-XTEN (FVII*) is intended to bypass the FIX and FVIII components of the intrinsic system when activated endogenously after administration.
  • FVII* inactive/low active form of FVII-XTEN
  • FIG. 9 is a graph of the distribution of cell cluster size (gray bars) and FVII ELISA titers in ng ml (black bars) by ELISA of clones from primary screening of pBC0014 CHO-K1 transformants (not all clones were labeled underneath the bars due to insufficient space)(see Example 25 for experimental details). Clones were sorted according to ELISA titer low to high (left to right).
  • FIG. 10 is a graph of cell counts (white bars) and FVII titers in ng/ml (black bars) of the top pBC0014 clones (see Example 25 for experimental details). Clones were sorted according to ELISA titer, low to high (left to right).
  • FIG. 11 is a graph of the ratio of FVII titer over cell count of the top pBC0014 clones (see Example 25 for experimental details). Clones were sorted according to the ratio, low to high (left to right).
  • FIG. 12 is a Western blot of top pBC0014 clones according to ELISA, clotting, ELISA/cell count and clotting/cell count ratios (see Example 25 for experimental details). Clone 6G1 expressed a truncated product and was not evaluated further.
  • FIG. 13 is a Western blot of the top pBC0016 clones according to ELISA, clotting, ELISA/cell count and clotting/cell count ratios (see Example 25 for experimental details).
  • FIG. 14 is a Western blot of the top pBC0018 clones according to ELISA, clotting, ELISA/cell count and clotting cell count ratios (see Example 25 for experimental details). Clone 3B2 expressed a truncated product and was not evaluated further.
  • FIG. 15 shows purification of FVII-AE864 by anti-GLA affinity chromatography (see Example 26 for experimental details). SDS-PAGE analysis demonstrating the purification of FVII-AE864 from concentrated supernatant and the >90% purity of the EDTA eluted fractions.
  • FIG. 16 shows activation of FVII-XTEN fusions to FVIIa-XTEN fusions by FXa treatment (see Example 26 for experimental details).
  • SDS-PAGE analysis demonstrates the appearance of a light chain band under reducing conditions after FXa treatment, but not in the untreated sample. Additionally, there is a downwards shift in the upper band indicating the loss of the light chain.
  • FIG. 17 shows an SDS-PAGE demonstrating auto-activation of FVII-XTEN fusions to FVIIa- XTEN fusions (see Example 26 for experimental details).
  • SDS-PAGE analysis demonstrating appearance of a light chain band under reducing conditions after FXa treatment and after incubation at 4°C at high concentration with CaCl 2 . Additionally, there is a downwards shift in the upper band indicating the loss of the light chain.
  • FIG. 18 shows SEC Analysis of FVII-AE864 and FVII-AE288 (see Example 26 for experimental details).
  • the SEC shows a monodispersed population with minimal contamination and no aggregates at the void volume of the column ( ⁇ 22 ml).
  • FIG. 19 shows the purification of FVII-AE864 by anion exchange chromatography (see Example 26 for experimental details).
  • the chromatograms depict the elution profiles of the total protein content and the FVII activity from a Macrocap Q column with the bulk of the activity eluting later than the contaminant proteins, creating a net 5-fold purification.
  • FIG. 20 shows purification of FVII-AE864 by hydrophobic interaction chromatography (see Example 26 for experimental details).
  • the chromatograms depict the elution profiles of the total protein content and the FVII activity from a toyopearl phenyl column with the bulk of the activity eluting earlier than the contaminant proteins, creating a net 2-fold purification
  • FIG. 21 shows two chromatography outputs demonstrating removal of aggregated protein from monomeric FVII-AE864 with anion exchange chromatography (see Example 26 for experimental details).
  • FIG. 21A is a chromatogram depicting the elution profile of FVII-XTEN from a macrocap Q column with two peaks eluting after the buffer related early peak.
  • FIG. 21B shows SEC chromatograms of the early and late macrocap Q peaks demonstrating the absence of aggregates in the early peak.
  • FIG. 22 shows results of ELISA or aPTT assays, showing FDi/cFXI/XTEN has enhanced activity compared to FEX-XTEN (see Example 29 for experimental details).
  • Transiently expressed FIX constructs were assayed for antigen content by ELISA and for activity by aPTT based assays. While the antigen content of FDi-XTEN was similar to the FEX/cFXI/XTEN constructs the activity was significantly increased. This increase is attributed to the specific action of the FXI protease in the assays as the FD /cTEV XTEN does not show a significantly different activity to FK-XTEN.
  • Note the ELISA titer of the FIX sample is 197 ng/ml and is off the scale of the graph.
  • FIG. 23 shows the pharmacokinetic profile after a single dose administered subcutaneously to rats, with the derived equivalent FVII concentration shown, as described in Example 30.
  • FIG. 24 shows the pharmacokinetic profile after a single dose administered subcutaneously to rats, with the derived equivalent FIX concentration shown, as described in Example 31.
  • FIG. 25 shows the pharmacokinetic profile (plasma concentrations) in cynomolgus monkeys after single doses of different compositions of GFP linked to unstructured polypeptides of varying length, administered either subcutaneously or intravenously, as described in Example 39.
  • the compositions were GFP-L288, GFP-L576, GFP-XTEN AF576, GFP-Y576 and XTEN AD836-GFP.
  • Blood samples were analyzed at various times after injection and the concentration of GFP in plasma was measured by ELISA using a polyclonal antibody against GFP for capture and a biotinylated preparation of the same polyclonal antibody for detection.
  • Results are presented as the plasma concentration versus time (h) after dosing and show, in particular, a considerable increase in half-life for the XTEN_AD836-GFP, the composition with the longest sequence length of XTEN.
  • the construct with the shortest sequence length, the GFP-L288 had the shortest half-life.
  • FIG. 26 shows an SDS-PAGE gel of samples from a stability study of the fusion protein of XTEN AE864 fused to the N-terminus of GFP (see Example 40).
  • the GFP-XTEN was incubated in cynomolgus plasma and rat kidney lysate for up to 7 days at 37°C.
  • GFP-XTEN administered to cynomolgus monkeys was also assessed. Samples were withdrawn at 0, 1 and 7 days and analyzed by SDS PAGE followed by detection using Western analysis with antibodies against GFP.
  • FIG. 27 shows three randomized libraries used for the third and fourth codons in the N-terminal sequences of clones from LCW546, LCW547 and LCW552 (see Example 14 for experimental details).
  • the libraries were designed with the third and fourth residues modified such that all combinations of allowable XTEN codons were present at these positions, as shown.
  • nine pairs of oligonucleotides encoding 12 amino acids with codon diversities of third and fourth residues were designed, annealed and ligated into the Ndel Bsal restriction enzyme digested stuffer vector pCW0551 (Stuffer-XTEN_AM875-GFP), and transformed into E. coli BL21Gold(DE3) competent cells to obtain colonies of the three libraries LCW0569, LCW0570, and LCW0571.
  • FIG. 28 shows a histogram of a retest of the top 75 clones after the optimization step, as described in Example 15, for GFP fluorescence signal, relative to the benchmark CBD AM875 construct. The results indicated that several clones were now superior to the benchmark clones.
  • FIG. 29 is a schematic of a combinatorial approach undertaken for the union of codon optimization preferences for two regions of the N-terminus 48 amino acids (see Example 16 for experimental details).
  • the approach created novel 48mers at the N-terminus of the XTEN protein for evaluation of the optimization of expression that resulted in leader sequences that can be a solution for the expression of XTEN proteins where the XTEN is N-terminal to the CF.
  • FIG. 30 shows an SDS-PAGE gel confirming the expression of preferred clones obtained from the XTEN N-terminal codon optimization experiments, in comparison to benchmark XTEN clones comprising CBD leader sequences at the N-terminus of the construct sequences, as described in Example 17.
  • FIG. 31 is a schematic flowchart of representative steps in the assembly, production and the evaluation of a XTEN.
  • FIG. 32 is a schematic flowchart of representative steps in the assembly of a CFXTEN polynucleotide construct encoding a fusion protein.
  • Individual oligonucleotides 501 are annealed into sequence motifs 502 such as a 12 amino acid motif ("12-mer"), which is subsequently ligated with an oligo containing Bbsl, and Kpnl restriction sites 503. Additional sequence motifs from a library are annealed to the 12-mer until the desired length of the XTEN gene 504 is achieved.
  • the XTEN gene is cloned into a stuffer vector.
  • the vector encodes an optional Flag sequence 506 followed by a stopper sequence that is flanked by Bsal, Bbsl, and Kpnl sites 507 and an FVII gene 508, resulting in the gene 500 encoding an XTEN-FVII fusion protein.
  • FIG. 33 is a schematic flowchart of representative steps in the assembly of a gene encoding fusion protein comprising a CF and XTEN, its expression and recovery as a fusion protein, and its evaluation as a candidate CFXTEN product.
  • FIG. 34 is a schematic representation of the design of CFXTEN expression vectors with different processing strategies.
  • FIG. 34A shows an expression vector encoding XTEN fused to the 3' end of the sequence encoding FVII. Note that no additional leader sequences are required in this vector.
  • FIG. 7B depicts an expression vector encoding XTEN fused to the 5' end of the sequence encoding FVII with a CBD leader sequence and a TEV protease site.
  • FIG. 7C depicts an expression vector as in FIG. 7B where WSGR Dkt. Ref. 32808-726601 the CBD and TEV processing sites have been replaced with an optimized N-terminal leader sequence (NTS).
  • FIG. 7D depicts an expression vector encoding an NTS sequence, an XTEN, a sequence encoding VFII, and than a second sequence encoding an XTEN.
  • FIG ' 35 shows results of a size exclusion chromatography analysis of glucagon-XTEN construct samples measured against protein standards of known molecular weight, with the graph output as absorbance versus retention volume, as described in Example 37.
  • the glucagon-XTEN constructs are 1) glucagon-Y288; 2) glucagonY-144; 3) glucagon-Y72; and 4) glucagon- Y36.
  • the results indicate an increase in apparent molecular weight with increasing length of XTEN moiety.
  • FIG. 36 shows sequence alignments between portions of native FIX, native FVII, and FVII-FDi sequence hybrids with different portions of the AP domain incorporated in the portion of the molecule spanning the EGF2 and Pro domains.
  • the legend provides construct names. Gaps in an individual sequence (dashes) represents stretches of non-homology to FIX but are otherwise continuous, linked sequences. The underlined amino acids are FD -derived sequence.
  • a cell includes a plurality of cells, including mixtures thereof.
  • polypeptide polypeptide
  • peptide protein
  • polymers of amino acids of any length may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified, for example, by disulfide bond formation, glycosylation, WSGR Dkt. Ref. 32808-726601 lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.
  • amino acid refers to either natural and/or unnatural or synthetic amino acids, including but not limited to both the D or L optical isomers, and amino acid analogs and peptidomimetics. Standard single or three letter codes are used to designate amino acids.
  • natural L-amino acid means the L optical isomer forms of glycine (G), proline (P), alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M), cysteine (C), phenylalanine (F), tyrosine (Y), tryptophan (W), histidine (H), lysine (K), arginine (R), glutamine (Q), asparagine (N), glutamic acid (E), aspartic acid (D), serine (S), and threonine (T).
  • non-naturally occurring means polypeptide or polynucleotide sequences that do not have a counterpart to, are not complementary to, or do not have a high degree of homology with a wild-type or naturally-occurring sequence found in a mammal.
  • a non-naturally occurring polypeptide or fragment may share no more than 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50% or even less amino acid sequence identity as compared to a natural sequence when suitably aligned.
  • hydrophilic and hydrophobic refer to the degree of affinity that a substance has with water.
  • a hydrophilic substance has a strong affinity for water, tending to dissolve in, mix with, or be wetted by water, while a hydrophobic substance substantially lacks affinity for water, tending to repel and not absorb water and tending not to dissolve in or mix with or be wetted by water.
  • Amino acids can be characterized based on their hydrophobicity. A number of scales have been developed.
  • hydrophilic amino acids are arginine, lysine, threonine, alanine, asparagine, and glutamine. Of particular interest are the hydrophilic amino acids aspartate, glutamate, and serine, and glycine.
  • hydrophobic amino acids are tryptophan, tyrosine, phenylalanine, methionine, leucine, isoleucine, and valine.
  • a “fragment” is a truncated form of a native biologically active protein that retains at least a portion of the therapeutic and/or biological activity.
  • a “variant” is a protein with sequence homology to the native biologically active protein that retains at least a portion of the therapeutic and/or biological activity of the biologically active protein.
  • a variant protein may share at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity compared with the reference biologically active protein.
  • biologically active protein moiety includes proteins modified deliberately, as for example, by site directed mutagenesis, insertions, or accidentally through mutations.
  • internal XTEN refers to XTEN sequences that have been inserted into the sequence of the coagulation factor.
  • Internal XTENs can be constructed by insertion of an XTEN sequence into the sequence of a coagulation factor such as FIX or FVII, either by insertion between two WSGR Dkt. Ref. 32808-726601 adjacent amino acids or domains of the coagulation factor or wherein XTEN replaces a partial, internal sequence of the coagulation factor.
  • terminal XTEN refers to XTEN sequences that have been fused to or in the N- or C-terminus of the coagulation factor or to a proteolytic cleavage sequence at the N- or C-terminus of the coagulation factor. Terminal XTENs can be fused to the native termini of the coagulation factor. Alternatively, terminal XTENs can replace a terminal sequence of the coagulation factor.
  • XTEN release site refers to a sequence in CFXTEN fusion proteins that can be recognized and cleaved by a mammalian protease, effecting release of an XTEN or a portion of an XTEN from the CFXTEN fusion protein.
  • mammalian protease means a protease that normally exists in the body fluids, cells or tissues of a mammal.
  • XTEN release sites can be engineered to be cleaved by various mammalian proteases (a.k.a.
  • XTEN release proteases such as FXIa, FXIIa, kallikrein, FVIIa, FEXa, FXa, FUa (thrombin), Elastase-2, MMP-12, MMP13, MMP-17, MMP-20, or any protease that is present during a clotting event.
  • Activity refers to retention of a biological activity of the native coagulation factor, wherein “biological activity” refers to an in vitro or in vivo biological function or effect, including but not limited to either receptor or ligand binding, enzymatic activity, or an effect on coagulation generally known in the art for the coagulation factor.
  • a "therapeutic effect” as applied to form(s) of a CFXTEN polypeptide provided herein refers to a physiologic effect, including but not limited to the curing, mitigation, reversal, amelioration or prevention of disease or conditions in humans or other animals, or to otherwise enhance physical or mental wellbeing of humans or animals.
  • a “therapeutically effective amount” means an amount of compound effective to prevent, alleviate, reverse or ameliorate symptoms of disease or a condition (e.g., a bleeding episode) or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
  • a "host cell” includes an individual cell or cell culture which can be or has been a recipient for the subject vectors.
  • Host cells include progeny of a single host cell. The progeny may not necessarily be completely identical (in morphology or in genomic of total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation.
  • a host cell includes cells transfected in vivo with a vector of this invention.
  • Isolated when used to describe the various polypeptides disclosed herein, means polypeptide that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. As is apparent to those of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, does not require "isolation" to distinguish it from its naturally occurring counterpart. In addition, a "concentrated”, WSGR Dkt. Ref. 32808-726601
  • polypeptide is distinguishable from its naturally occurring counterpart in that the concentration or number of molecules per volume is generally greater than that of its naturally occurring counterpart.
  • a polypeptide made by recombinant means and expressed in a host cell is considered to be “isolated.”
  • An "isolated" polynucleotide or polypeptide-encoding nucleic acid or other polypeptide-encoding nucleic acid is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the polypeptide- encoding nucleic acid.
  • An isolated polypeptide-encoding nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated polypeptide-encoding nucleic acid molecules therefore are distinguished from the specific polypeptide-encoding nucleic acid molecule as it exists in natural cells.
  • an isolated polypeptide-encoding nucleic acid molecule includes polypeptide-encoding nucleic acid molecules contained in cells that ordinarily express the polypeptide where, for example, the nucleic acid molecule is in a chromosomal or extra-chromosomal location different from that of natural cells.
  • a "chimeric" protein contains at least one fusion polypeptide comprising regions in a different position in the sequence than that which occurs in nature.
  • the regions may normally exist in separate proteins and are brought together in the fusion polypeptide; or they may normally exist in the same protein but are placed in a new arrangement in the fusion polypeptide.
  • a chimeric protein may be created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship.
  • Conjugated refers to the joining together of two or more chemical elements or components, by whatever means including chemical conjugation or recombinant means.
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence.
  • operably linked means that the DNA sequences being linked are contiguous, and in reading phase or in-frame.
  • An "in-frame fusion” refers to the joining of two or more open reading frames (ORFs) to form a continuous longer ORF, in a manner that maintains the correct reading frame of the original ORFs.
  • ORFs open reading frames
  • the resulting recombinant fusion protein is a single protein containing two or more segments that correspond to polypeptides encoded by the original ORFs (which segments are not normally so joined in nature).
  • a “linear sequence” or a “sequence” is an order of amino acids in a polypeptide in an amino to carboxyl terminus direction in which residues that neighbor each other in the sequence are contiguous in the primary structure of the polypeptide.
  • a “partial sequence” is a linear sequence of part of a polypeptide that is known to comprise additional residues in one or both directions.
  • Heterologous means derived from a genotypically distinct entity from the rest of the entity to which it is being compared.
  • a glycine rich sequence removed from its native coding sequence and operatively linked to a coding sequence other than the native sequence is a heterologous glycine rich sequence.
  • heterologous as applied to a polynucleotide, a polypeptide, means that WSGR Dkt Ref. 32808-726601 the polynucleotide or polypeptide is derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared.
  • polynucleotides refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The following are non-limiting examples of
  • polynucleotides coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer.
  • the sequence of nucleotides may be interrupted by non-nucleotide components.
  • a polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
  • complement of a polynucleotide denotes a polynucleotide molecule having a complementary base sequence and reverse orientation as compared to a reference sequence, such that it could hybridize with a reference sequence with complete fidelity.
  • Recombinant as applied to a polynucleotide means that the polynucleotide is the product of various combinations of in vitro cloning, restriction and/or ligation steps, and other procedures that result in a construct that can potentially be expressed in a host cell.
  • gene and “gene fragment” are used interchangeably herein. They refer to a polynucleotide containing at least one open reading frame that is capable of encoding a particular protein after being transcribed and translated.
  • a gene or gene fragment may be genomic or cDNA, as long as the polynucleotide contains at least one open reading frame, which may cover the entire coding region or a segment thereof.
  • a “fusion gene” is a gene composed of at least two heterologous polynucleotides that are linked together.
  • Homology refers to sequence similarity or interchangeability between two or more polynucleotide sequences or two or more polypeptide sequences.
  • BestFit a program such as BestFit to determine sequence identity, similarity or homology between two different amino acid sequences
  • the default settings may be used, or an appropriate scoring matrix, such as blosum45 or blosum80, may be selected to optimize identity, similarity or homology scores.
  • an appropriate scoring matrix such as blosum45 or blosum80
  • polynucleotides that are homologous are those which hybridize under stringent conditions as defined herein and have at least 70%, preferably at least 80%, more preferably at least 90%, more preferably 95%, more preferably 97%, more preferably 98%, and even more preferably 99% sequence identity compared to those sequences.
  • “Ligation” refers to the process of forming phosphodiester bonds between two nucleic acid fragments or genes, linking them together.
  • the ends of WSGR Dkt. Ref. 32808-726601 the DNA must be compatible with each other. In some cases, the ends will be directly compatible after endonuclease digestion. However, it may be necessary to first convert the staggered ends commonly produced after endonuclease digestion to blunt ends to make them compatible for ligation.
  • stringent conditions or “stringent hybridization conditions” includes reference to conditions under which a polynucleotide will hybridize to its target sequence, to a detectably greater degree than other sequences (e.g., at least 2-fold over background).
  • stringency of a polynucleotide to a detectably greater degree than other sequences (e.g., at least 2-fold over background).
  • hybridization is expressed, in part, with reference to the temperature and salt concentration under which the wash step is carried out.
  • stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short polynucleotides (e.g., 10 to 50 nucleotides) and at least about 60°C for long polynucleotides (e.g., greater than 50 nucleotides)— for example, "stringent conditions" can include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37°C, and three washes for 15 min each in 0.1 *SSC/1% SDS at 60°C to 65°C.
  • temperatures of about 65°C, 60°C, 55°C, or 42°C may be used.
  • SSC concentration may be varied from about 0.1 to 2 ⁇ SSC, with SDS being present at about 0.1%.
  • wash temperatures are typically selected to be about 5°C to 20°C lower than the thermal melting point for the specific sequence at a defined ionic strength and pH.
  • the Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • blocking reagents are used to block non-specific hybridization.
  • blocking reagents include, for instance, sheared and denatured salmon sperm DNA at about 100-200 ⁇ g ml.
  • Organic solvent such as formamide at a concentration of about 35-50% v/v
  • RNA:DNA hybridizations Useful variations on these wash conditions will be readily apparent to those of ordinary skill in the art.
  • percent identity and % identity refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.
  • Percent identity may be measured over the length of an entire defined polynucleotide sequence, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polynucleotide sequence, for instance, a fragment of at least 45, at least 60, at least 90, at least 120, at least 150, at least 210 or at least 450 contiguous residues.
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
  • Percent (%) sequence identity is defined as the percentage of amino acid residues in a query sequence that are identical with the amino acid residues of a second, reference polypeptide sequence or a portion thereof, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.
  • Percent identity may be measured over the length of an entire defined polypeptide sequence, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues.
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
  • non-repetitiveness refers to a lack or limited degree of internal homology in a peptide or polypeptide sequence.
  • substantially non- repetitive can mean, for example, that there are few or no instances of four contiguous amino acids in the sequence that are identical amino acid types or that the polypeptide has a subsequence score (defined infra) of 10 or less or that there isn't a pattern in the order, from N- to C-terminus, of the sequence motifs that constitute the polypeptide sequence.
  • a “repetitiveness” as used herein in the context of a polypeptide refers to the degree of internal homology in a peptide or polypeptide sequence.
  • a “repetitive” sequence may contain multiple identical copies of short amino acid sequences.
  • a polypeptide sequence of interest may be divided into n-mer sequences and the number of identical sequences can be counted.
  • Highly repetitive sequences contain a large fraction of identical sequences while non-repetitive sequences contain few identical sequences.
  • a sequence can contain multiple copies of shorter sequences of defined or variable length, or motifs, in which the motifs themselves have non-repetitive sequences, rendering the full-length polypeptide substantially non-repetitive.
  • the length of polypeptide within which the non-repetitiveness is measured can vary from 3 amino acids to about 200 amino acids, about from 6 to about 50 amino acids, or from about 9 to about 14 amino acids.
  • “Repetitiveness” used in the context of polynucleotide sequences refers to the degree of internal homology in the sequence such as, for example, the frequency of identical nucleotide sequences of a given length. Repetitiveness can, for example, be measured by analyzing the frequency of identical sequences.
  • a "vector” is a nucleic acid molecule, preferably self-replicating in an appropriate host, which transfers an inserted nucleic acid molecule into and/or between host cells.
  • the term includes vectors that WSGR Dkt. Ref. 32808-726601 function primarily for insertion of DNA or RNA into a cell, replication of vectors that function primarily for the replication of DNA or RNA, and expression vectors that function for transcription and/or translation of the DNA or RNA. Also included are vectors that provide more than one of the above functions.
  • An “expression vector” is a polynucleotide which, when introduced into an appropriate host cell, can be transcribed and translated into a polypeptide(s).
  • An "expression system” usually connotes a suitable host cell comprised of an expression vector that can function to yield a desired expression product.
  • serum degradation resistance refers to the ability of the polypeptides to withstand degradation in blood or components thereof, which typically involves proteases in the serum or plasma.
  • the serum degradation resistance can be measured by combining the protein with human (or mouse, rat, monkey, as appropriate) serum or plasma, typically for a range of days (e.g. 0.25, 0.5, 1, 2, 4, 8, 16 days), typically at about 37°C.
  • the samples for these time points can be run on a Western blot assay and the protein is detected with an antibody.
  • the antibody can be to a tag in the protein. If the protein shows a single band on the western, where the protein's size is identical to that of the injected protein, then no degradation has occurred.
  • the time point where 50% of the protein is degraded is the serum degradation half-life or "serum half-life" of the protein.
  • t as used herein means the terminal half-life calculated as ln(2)/K d .
  • K « i is the terminal elimination rate constant calculated by linear regression of the terminal linear portion of the log concentration vs. time curve.
  • Half-life typically refers to the time required for half the quantity of an administered substance deposited in a living organism to be metabolized or eliminated by normal biological processes.
  • terminal half-life “elimination half-life” and “circulating half- life” are used interchangeably herein.
  • Active clearance means the mechanisms by which CF is removed from the circulation other than by filtration or coagulation, and which includes removal from the circulation mediated by cells, receptors, metabolism, or degradation of the CF.
  • Apparent molecular weight factor and "apparent molecular weight” are related terms referring to a measure of the relative increase or decrease in apparent molecular weight exhibited by a particular amino acid sequence.
  • the apparent molecular weight is determined using size exclusion chromatography (SEC) and similar methods compared to globular protein standards and is measured in "apparent kD" units.
  • the apparent molecular weight factor is the ratio between the apparent molecular weight and the actual molecular weight; the latter predicted by adding, based on amino acid composition, the calculated molecular weight of each type of amino acid in the composition or by estimation from comparison to molecular weight standards in an SDS electrophoresis gel.
  • hydrodynamic radius or "Stokes radius” is the effective radius (R h in nm) of a molecule in a solution measured by assuming that it is a body moving through the solution and resisted by the solution's viscosity.
  • the hydrodynamic radius measurements of WSGR Dkt. Ref. 32808-726601 the XTEN fusion proteins correlate with the 'apparent molecular weight factor', which is a more intuitive measure.
  • the "hydrodynamic radius” of a protein affects its rate of diffusion in aqueous solution as well as its ability to migrate in gels of macromolecules.
  • the hydrodynamic radius of a protein is determined by its molecular weight as well as by its structure, including shape and compactness. Methods for determining the hydrodynamic radius are well known in the art, such as by the use of size exclusion chromatography (SEC), as described in U.S. Patent Nos. 6,406,632 and 7,294,513. Most proteins have globular structure, which is the most compact three-dimensional structure a protein can have with the smallest hydrodynamic radius. Some proteins adopt a random and open, unstructured, or 'linear' conformation and as a result have a much larger hydrodynamic radius compared to typical globular proteins of similar molecular weight.
  • SEC size exclusion chromatography
  • Physiological conditions refers to a set of conditions in a living host as well as in vitro conditions, including temperature, salt concentration, pH, that mimic those conditions of a living subject.
  • a host of physiologically relevant conditions for use in in vitro assays have been established.
  • a physiological buffer contains a physiological concentration of salt and is adjusted to a neutral pH ranging from about 6.5 to about 7.8, and preferably from about 7.0 to about 7.5.
  • a variety of physiological buffers are listed in Sambrook et al. (1989).
  • Physiologically relevant temperature ranges from about 25°C to about 38°C, and preferably from about 35°C to about 37°C.
  • a "reactive group” is a chemical structure that can be coupled to a second reactive group.
  • reactive groups are amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups, aldehyde groups, azide groups. Some reactive groups can be activated to facilitate coupling with a second reactive group. Non-limiting examples for activation are the reaction of a carboxyl group with carbodiimide, the conversion of a carboxyl group into an activated ester, or the conversion of a carboxyl group into an azide function.
  • Controlled release agent “slow release agent”, “depot formulation” and “sustained release agent” are used interchangeably to refer to an agent capable of extending the duration of release of a polypeptide of the invention relative to the duration of release when the polypeptide is administered in the absence of agent.
  • Different embodiments of the present invention may have different release rates, resulting in different therapeutic amounts.
  • antigen binds to or has specificity against.
  • target antigen binds to or has specificity against.
  • payload refers to a protein or peptide sequence that has biological or therapeutic activity; the counterpart to the pharmacophore of small molecules.
  • payloads include, but are not limited to, cytokines, enzymes, hormones and blood and growth factors.
  • Payloads can further comprise genetically fused or chemically conjugated moieties such as chemotherapeutic agents, antiviral compounds, toxins, or contrast agents. These conjugated moieties can be joined to the rest of the polypeptide via a linker that may be cleavable or non-cleavable.
  • antagonist includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of a native polypeptide disclosed herein.
  • Methods for identifying antagonists of a polypeptide may comprise contacting a native polypeptide with a candidate antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the native polypeptide.
  • antagonists may include proteins, nucleic acids, carbohydrates, antibodies or any other molecules that decrease the effect of a biologically active protein.
  • agonist is used in the broadest sense and includes any molecule that mimics a biological activity of a native polypeptide disclosed herein. Suitable agonist molecules specifically include agonist antibodies or antibody fragments, fragments or amino acid sequence variants of native polypeptides, peptides, small organic molecules, etc. Methods for identifying agonists of a native polypeptide may comprise contacting a native polypeptide with a candidate agonist molecule and measuring a detectable change in one or more biological activities normally associated with the native polypeptide.
  • Activity refers to an action or effect of a component of a fusion protein consistent with that of the corresponding native biologically active protein, wherein “biological activity” refers to an in vitro or in vivo biological function or effect, including but not limited to receptor binding, antagonist activity, agonist activity, or a cellular or physiologic response.
  • treatment or “treating,” or “palliating” or “ameliorating” is used
  • compositions may be administered to a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made.
  • a "therapeutic effect”, as used herein, refers to a physiologic effect, including but not limited to the cure, mitigation, amelioration, or prevention of disease in humans or other animals, or to otherwise enhance physical or mental wellbeing of humans or animals, caused by a fusion polypeptide of the invention other than the ability to induce the production of an antibody against an antigenic epitope possessed by the biologically active protein. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
  • terapéuticaally effective amount refers to an amount of a biologically active protein, either alone or as a part of a fusion protein WSGR Dkt. Ref. 32808-726601 composition, that is capable of having any detectable, beneficial effect on any symptom, aspect, measured parameter or characteristics of a disease state or condition when administered in one or repeated doses to a subject. Such effect need not be absolute to be beneficial.
  • terapéuticaally effective dose regimen refers to a schedule for consecutively administered multiple doses (i.e., at least two or more) of a biologically active protein, either alone or as a part of a fusion protein composition, wherein the doses are given in therapeutically effective amounts to result in sustained beneficial effect on any symptom, aspect, measured parameter or characteristics of a disease state or condition.
  • coagulation factor refers to factor IX (FIX), factor VII (FVII), sequence combinations of FVII and FIX, or mimetics, sequence variants and truncated versions thereof, (a) Factor IX
  • Fractor IX or "FIX” means a coagulation factor protein and species and sequence variants thereof, and includes, but is not limited to, the 461 single-chain amino acid sequence of human FIX precursor polypeptide ("prepro") and the 415 single-chain amino acid sequence of mature human FIX.
  • FIX inlcudes any form of factor IX molecule with the typical characteristics of blood coagulation factor IX.
  • factor IX and FIX are intended to encompass polypeptides that comprise the domains Gla (region containingy-carboxyglutamic acid residues), EGF1 and EGF2 (region containing sequences homologous to human epidermal growth factor), activation peptide (formed by residues R136- R180 of the mature FIX), and the C-terminal protease domain ("Pro"), or synonyms of these domains known in the art, or can be a truncated fragment or a sequence variant that retains at least a portion of the biological activity of the native protein.
  • FIX or sequence variants have been cloned, as described in U.S. Patent Nos. 4,770,999, 7,700,734, and cDNA coding for human factor IX has been isolated,
  • Human factor DC is encoded by a single-copy gene residing on the X-chromosome at q27.1.
  • the human FIX mRNA is composed of 205 bases for the 5' untranslated region, 1383 bases for the prepro factor ⁇ , a stop codon and 1392 bases for the 3' untranslated region.
  • the FIX polypeptide is 55 kDa, synthesized as a prepropolypetide chain composed of three regions: a signal peptide of 28 amino acids, a propeptide of 18 amino acids, which is required for gamma-carboxylation of glutamic acid residues, and a mature factor ⁇ of 415 amino acids.
  • the propeptide is an 18-amino acid residue sequence N-terminal to the gamma-carboxyglutamate domain.
  • the propeptide binds vitamin independent gamma carboxylase and then is cleaved from the precursor polypeptide of FIX by an endogenous protease, most likely PACE (paired basic amino acid cleaving enzyme), also known as furin or PCSK3. Without the gamma carboxylation, the Gla domain is unable to bind calcium to assume the correct conformation necessary to anchor the protein to negatively charged phospholipid surfaces, thereby rendering factor IX nonfunctional.
  • PACE paired basic amino acid cleaving enzyme
  • the Gla domain also depends on cleavage of the propeptide for proper function, since retained propeptide interferes with conformational changes of the Gla domain necessary for optimal binding to calcium and phospholipid.
  • the resulting mature factor DC is secreted by liver cells into the blood stream as an inactive zymogen, a single chain protein of 415 amino acid residues that contains approximately 17% carbohydrate by weight (Schmidt, A. E., et al. (2003) Trends Cardiovasc Med, 13: 39).
  • the mature factor DC is composed of several domains that in an N- to C-terminus configuration are: a Gla domain, an EGF1 domain, an EGF2 domain, an activation peptide (AP) domain, and a protease (or catalytic) domain.
  • FDC contains two activation peptides formed by R145-A146 and R180-V181, respectively. Following activation, the single-chain FDC becomes a 2-chain molecule, in which the two chains are linked by a disulfide bond attaching the enzyme to the Gla domain.
  • CFs can be engineered by replacing their activation peptides resulting in altered activation specificity.
  • mature FDC must be activated by activated factor XI to yield factor DCa.
  • the protease domain provides, upon activation of FIX to FDCa, the catalytic activity of FDC.
  • Activated factor VIII (F Villa) is the specific cofactor for the full expression of FDCa activity.
  • Proteins involved in clotting include factor I, factor ⁇ , factor m, factor IV, factor V, factor VI, factor VII, factor VIII, factor DC, factor X, factor XI, factor XII, factor XIII, Protein C, and tissue factor ("clotting proteins").
  • the majority of the clotting proteins is present in zymogen form that when activatedexhibits a pro-coagulant protease activity to activate other clotting proteins, contributing to the intrinsic or extrinsic coagulation parthway and clot formation.
  • FDC associates with a complex of activated factor VIII, factor X, calcium, and phospholipid. In the complex, FDC is activated by factor XIa.
  • factor DC The activation of factor DC is achieved by a two-step removal of the activation peptide (Ala 146 -Arg 180) from the molecule (Bajaj et al., Human factor DC and factor DCa, in METHODS DM ENZYMOLOGY. 1993).
  • the first cleavage is made at the Arg 145 - Ala 146 site by either factor XIa or factor Vila/tissue factor.
  • the second and rate limiting cleavage is WSGR Dkt Ref. 32808-726601 made at Arg 180 -Val 181.
  • the activation removes 35 residues.
  • Activated human factor EX exists as a heterodimer of the C-terminal heavy chain (28 kDa) and an N-terminal light chain (18 kDa), which are held together by one disulfide bridge attaching the enzyme to the Gla domain.
  • Factor EXa in turn activates factor X in concert with activated factor VIII.
  • factors IX and X can both be activated by factor Vila complexed with lipidated Tissue Factor, generated via the extrinsic pathway.
  • Factor Xa then participates in the final common pathway whereby prothrombin is converted to thrombin, and thrombin in turn converts fibrinogen to fibrin to form the clot.
  • Replacement therapy with these proteins may be used in the therapeutic intervention of hemophilia B (Christmas Disease) and factor DC-related bleeding disorders.
  • Factor DC can be used in the treatment of both conditions. In some cases, however, patients develop antibodies against the administered proteins that reduce or negate the efficacy of the treatment.
  • the invention contemplates inclusion of FDC sequences in the CFXTEN compositions that have homology to FDC sequences, sequence fragments that are natural, such as from humans, non-human primates, mammals (including domestic animals), and non-natural sequence variants which retain at least a portion of the biologic activity or biological function of FDC and/or that are useful for preventing, treating, mediating, or ameliorating a coagulation factor-related disease, deficiency, disorder or condition (e.g., bleeding episodes related to trauma, surgery, of deficiency of a coagulation factor). Sequences with homology to human FEX can be found by standard homology searching techniques, such as NCBI BLAST.
  • the FDC incorporated into the subject compositions is a recombinant polypeptide with a sequence corresponding to a protein found in nature.
  • the FDC is a sequence variant, fragment, homolog, or a mimetics of a natural sequence that retains at least a portion of the biological activity of the corresponding native FDC.
  • Table 1 provides a non-limiting list of amino acid sequences of FIX that are encompassed by the CFXTEN fusion proteins of the invention. Any of the FDC sequences or homologous derivatives to be incorporated into the fusion protein compositions can be constructed by shuffling individual mutations between the amino acid sequences of Table 1 and evaluated for activity.
  • FDC that can be incorporated into a CFXTEN fusion protein includes a protein that has at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to an amino acid sequence selected from Table 1.
  • Factor VII or “FVII” means a coagulation factor protein and species and sequence variants thereof, and includes, but is not limited to, both the inactive and activated forms (unless indicated to the contrary) of the 406 single-chain amino acid sequence of human FVII, and the 444 amino acid sequence of the precursor protein.
  • factor VII and FVII encompass polypeptides that comprise the domains Gla (region containing ⁇ -carboxyglutamic acid residues), EGF 1 and EGF2 (region containing sequences homologous to human epidermal growth factor), an activation peptide domain that spans the sequence between the EGF2 and Pro domains, and a catalytic or peptidase SI domain ("Pro" region containing the serine protease catalytic triad), or synonyms of these domains known in the art, or can be a WSGR Dkt. Ref. 32808-726601 truncated fragment or a sequence variant that retains at least a portion of the biological activity of the native protein.
  • Factor VII a vitamin K-dependent plasma protein produced by the liver, initially circulates in the blood as a zymogen.
  • the main role of factor VII is to initiate the process of coagulation in conjunction with tissue factor (TF).
  • tissue factor Upon vessel injury, tissue factor is exposed to the blood and circulating factor VII.
  • FVII is activated to become the activated form of factor VII (FVIIa) by different proteases, among which are thrombin (factor Ila), factor Xa, IXa, Xlla, and the FVIIa-TF complex itself.
  • FVII zymogen is activated by proteolytic cleavage at a single site, Arg 152 - He 153 , resulting in a two-chain protease linked by a single disulphide bond (FVIIa).
  • FVIIa binds its cofactor, tissue factor (TF), to form a complex which can activate factor X (FX) to FXa, thereby initiating a coagulation cascade that results in fibrin formation and hemostasis.
  • tissue factor (TF) tissue factor
  • FX factor X
  • the complete nucleotide and amino acid sequences for human factor VII are known, and human FVII or sequence variants have been cloned, as described in U.S. Patent Nos. 4,784,950, 5,833,982, 6911323, and 7,026,524.
  • FVII also is utilized in connection with treatment of uncontrolled bleedings, such as trauma, and it is believed that factor Vila is capable of activating factor X to factor Xa without binding to tissue factor, and this activation reaction is believed to occur primarily on activated blood platelets (Hedner et al. Blood Coagulation & Fibrinolysis, 2000; 11;107-11 1).
  • Sequence variants of factor VII include, polypeptides having an amino acid sequence that differs from the sequence of wild-type factor VII by insertion, deletion, or substitution of one or more amino acids.
  • FVII variants are known in the art, including those described in United States Patent and Application Nos. 6,960,657, 7,176,288, 7414022, 7,700,733, 20060205036A1, 20080318276A1, and 20090011992A1, which are incorporated herein by reference.
  • Recombinant FVIIa has been approved for the treatment of hemophilia A or B patients that have inhibitors to FVIII or FIX, and also is used to stop bleeding episodes or prevent bleeding associated with trauma and/or surgery.
  • Recombinant FVIIa also has been approved for the treatment of patients with congenital FVII deficiency, and is increasingly being utilized in off-label uses, such as the treatment of bleeding associated with other congenital or acquired bleeding disorders, trauma, and surgery in hemophilic and non-hemophilic patients.
  • the invention contemplates inclusion in the CFXTEN compositions sequences with homology to FVII sequences, sequence fragments, mimetics and non-natural sequence variants which retain at least WSGR Dkt. Ref. 32808-726601 a portion of the biologic activity or biological function of FVIIa that are useful for preventing, treating, mediating, or ameliorating a CF-related disease, deficiency, disorder or condition.
  • compositions comprising the inactive form of FVII that can be activated by mammalian endogenous proteases (described more fully below) or undergo autoactivation represents a means to treat subjects with certain forms of chronic coagulopathies with what is essentially a "prodrug" form of FVII.
  • Table 2 provides a list of sequences of FVII that are encompassed by the CFXTEN fusion proteins of the invention. FVII sequences or homologous derivatives constructed by shuffling individual mutations between species or families that retain at least a portion of the biological activity of the native CF are useful for the fusion proteins of this invention.
  • FVII that can be incorporated into a CFXTEN fusion protein include a protein that exhibits at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to a sequence selected from Table 2.
  • the present invention provides fusion protein compositions comprising coagulation factors (CF).
  • CF coagulation factors
  • One way to increase the circulation half-life of a therapeutic protein is to reduce the renal clearance of the protein. This may be achieved by conjugating the protein to a polymer that s capable of conferring an increased molecular size (or hydrodynamic radius) to the protein, and hence, reduces renal clearance.
  • one object of the present invention is to provide improved FIX or FVII (or FVIIa) molecules with a longer circulation, or terminal half-life (thereby decreasing the number of necessary administrations) and that retain at least a portion of the activity of the native coagulation factors, thereby to treat coagulation WSGR Dkt. Ref. 32808-726601 deficiencies and uncontrolled bleedings more efficiently.
  • the invention provides isolated monomelic fusion proteins of CF comprising the full-length sequence or sequence variants of a CF, such as FIX or FVII, covalently linked to extended recombinant polypeptides ("XTEN” or "XTENs").
  • XTEN extended recombinant polypeptides
  • the fusion proteins optionally include spacer sequences that further comprise cleavage sequences to release the CF from the fusion protein when acted on by a protease.
  • the invention provides an isolated fusion protein comprising at least a first biologically active coagulation factor protein covalently linked to one or more extended recombinant polypeptides ("XTEN"), resulting in a fusion protein composition (hereinafter "CFXTEN").
  • XTEN extended recombinant polypeptides
  • CFXTEN fusion protein composition
  • the term "CFXTEN”, as used herein, is meant to encompass fusion polypeptides that comprise one or more payload regions each comprising a biologically active CF that mediates one or more biological or therapeutic activities associated with a coagulation factor and at least one other region comprising at least a first XTEN polypeptide that serves as a carrier.
  • the coagulation factor is FIX or a sequence variant of FIX, as disclosed above (including sequences with homology to the sequences of Table 1).
  • the coagulation factor is FVII, which can include the activated form of FVII, or a sequence variant of FVII, as disclosed above (including sequences with homology with the sequences of Table 2).
  • activation of the FVII component may be carried out by exposure to activated factor X, by auto- activation, or according to procedures known in the art, such as those disclosed by Osterud, et al., Biochemistry 11 :2853-2857 (1972); Thomas, U.S. Pat. No.
  • factor VII can be activated by passing it through an ion-exchange chromatography column (see, e.g., Bjoern et al. Research Disclosure (1986) 269:564-565), such as Mono Q (Pharmacia fine Chemicals) or similar chromatography resins.
  • the CF of the subject compositions are well known in the art and descriptions and sequences are available in public databases such as Chemical Abstracts Services Databases (e.g., the CAS Registry), GenBank, The Universal Protein Resource (UniProt) and subscription provided databases such as GenSeq (e.g., Derwent).
  • Chemical Abstracts Services Databases e.g., the CAS Registry
  • GenBank GenBank
  • UniProt Universal Protein Resource
  • GenSeq e.g., Derwent
  • Polynucleotide sequences may be a wild type polynucleotide sequence encoding a given CF (e.g., either full length or mature), or in some instances the sequence may be a variant of the wild type polynucleotide sequence (e.g., a polynucleotide which encodes the wild type biologically active protein, wherein the DNA sequence of the polynucleotide has been optimized, for example, for expression in a particular species; or a polynucleotide encoding a variant of the wild type protein, such as a site directed mutant or an allelic variant.
  • a variant of the wild type protein e.g., a polynucleotide which encodes the wild type biologically active protein, wherein the DNA sequence of the polynucleotide has been optimized, for example, for expression in a particular species
  • a polynucleotide encoding a variant of the wild type protein such as a site directed mutant or an allelic variant
  • the CF for inclusion in the CFXTEN of the invention include coagulation factors or sequence variants that are useful, when administered to a subject, for mediating or preventing or ameliorating a disease, disorder or condition associated with bleeding disorders, coagulation factor deficiencies or defects in a coagulation factor.
  • coagulation factors or sequence variants that are useful, when administered to a subject, for mediating or preventing or ameliorating a disease, disorder or condition associated with bleeding disorders, coagulation factor deficiencies or defects in a coagulation factor.
  • CFXTEN fusion protein compositions for which an increase in a pharmacokinetic parameter, increased solubility, increased stability, or some other enhanced pharmaceutical property compared to native CF is sought, or for which increasing the terminal half-life would improve efficacy, safety, or result in reduced dosing frequency and/or improve patient compliance.
  • the CFXTEN fusion protein compositions are prepared with various objectives in mind, including improving the therapeutic efficacy of the bioactive CF by, for example, increasing the in vivo exposure or the length that the CFXTEN remains within the therapeutic window when administered to a subject, compared to a CF not linked to XTEN.
  • the CF incorporated into the subject compositions can be a recombinant polypeptide with a sequence corresponding to a protein found in nature.
  • the CF is a sequence variant, fragment, homolog, or mimetic of a natural sequence that retain at least a portion of the biological activity of the native CF.
  • a CF is a sequence that exhibits at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99%, or 100% sequence identity compared to a protein sequence selected from Table 1 or from Table 2.
  • a CFXTEN fusion protein comprises a single CF molecule linked to a single XTEN (e.g., an XTEN as described more fully below).
  • the CFXTEN comprises a first CF and a second molecule of the same CF, resulting in a fusion protein comprising the two CF linked to one or more XTEN in an N- to C- terminus configuration selected from Table 6.
  • the CFXTEN fusion protein comprises a single CF molecule linked to a first and a second XTEN, in which the CF is a sequence that exhibits at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99%, or 100% sequence identity compared to a protein sequence selected from Table 1 or from Table 2, and the first and/or the second XTEN are sequences that exhibits at least about 80% sequence identity, or alternatively 81 , 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%
  • the subject CFXTEN of the present invention exhibits an enhancement of one or more pharmacokinetic parameters compared to the native CF.
  • the CFXTEN with enhanced pharmacokinetic parameters permits less frequent dosing or an enhanced pharmacologic effect, inlcuding but not limited to maintaining the biologically active CFXTEN within the therapeutic window between the minimum effective dose or blood concentration (C m j n ) and the maximum tolerated dose or blood concentration (Cniax) for a longer period of time compared to the CF not linked to XTEN.
  • the linking of the CF to a fusion protein comprising a select XTEN sequence(s) can result in an improvement in these properties, making them more useful as therapeutic or preventive agents compared to CF not linked to WSGR Dkt Ref. 32808-726601
  • the subject CFXTEN of the present invention has a cleavage sequence incorporated between the CF and the XTEN and the biologic activity of the CF component is enhanced by the release of the CF from the fusion protein by cleavage of the cleavage sequence by an endogenous protease, as described below.
  • the invention provides XTEN polypeptide compositions that are useful as a fusion protein partner to which CF is linked, resulting in a CFXTEN fusion protein.
  • XTEN are generally extended length polypeptides with non-naturally occurring, substantially non-repetitive sequences that are composed mainly of small hydrophilic amino acids, with the sequence having a low degree or no secondary or tertiary structure under physiologic conditions.
  • XTENs have utility as a fusion protein partners in that they serve as a "carrier,” conferring certain desirable pharmacokinetic, physicochemical and pharmaceutical properties when linked to a CF protein to a create a fusion protein. Such desirable properties include but are not limited to enhanced pharmacokinetic parameters and solubility characteristics of the compositions, amongst other properties described herein. Such fusion protein compositions have utility to treat certain coagulation factor-related diseases, disorders or conditions, as described herein. As used herein, "XTEN” specifically excludes whole antibodies or antibody fragments (e.g. single-chain antibodies and Fc fragments).
  • the XTEN is a long polypeptide having greater than about 100 to about 3000 amino acid residues when used as a carrier or greater than 400 to about 3000 residues cumulatively when more than one XTEN unit is used in a single fusion protein.
  • an XTEN sequence shorter than 100 amino acid residues such as about 96, or about 84, or about 72, or about 60, or about 48, or about 36 amino acid residues are incorporated into a fusion protein composition with the CF to effect the property.
  • the selection criteria for the XTEN to be linked to the biologically active proteins used to create the inventive fusion proteins compositions generally relate to attributes of physical/chemical properties and conformational structure of the XTEN that is, in turn, used to confer enhanced pharmaceutical and pharmacokinetic properties to the fusion proteins compositions.
  • the XTEN of the present invention exhibits one or more of the following advantageous properties: conformational flexibility, enhanced aqueous solubility, high degree of protease resistance, low immunogenicity, low binding to mammalian receptors, and increased hydrodynamic (or Stokes) radii; properties that make them particularly useful as fusion protein partners.
  • Non-limiting examples of the properties of the fusion proteins comprising CF that are enhanced by XTEN include increases in the overall solubility and/or metabolic stability, reduced susceptibility to proteolysis, reduced immunogenicity, reduced rate of absorption when administered subcutaneously or intramuscularly, and enhanced pharmacokinetic WSGR Dkt Ref.
  • 32808-726601 properties such as longer terminal half-life and increased area under the curve (AUC), slower absorption after subcutaneous or intramuscular injection (compared to CF not linked to XTEN and administered by a similar route) such that the Cmax is lower, which, in turn, results in reductions in adverse effects of the CF that, collectively, results in an increased period of time that a fusion protein of a CFXTEN composition administered to a subject retains therapeutic activity.
  • AUC area under the curve
  • a variety of methods and assays are known in the art for determining the physical/chemical properties of proteins such as the compositions comprising the inventive XTEN. Such properties inlcude but are not limited to secondary or tertiary structure, solubility, protein aggregation, melting properties, contamination and water content. Such methods include analytical centrifugation, EPR, HPLC-ion exchange, HPLC-size exclusion, HPLC-reverse phase, light scattering, capillary electrophoresis, circular dichroism, differential scanning calorimetry, fluorescence, HPLC-ion exchange, HPLC-size exclusion, IR, NMR, Raman spectroscopy, refractometry, and UV/Visible spectroscopy. Additional methods are disclosed in Arnau, et al, Prot Expr and Purif (2006) 48, 1-13.
  • XTEN is designed to behave like denatured peptide sequence under physiological conditions, despite the extended length of the polymer.
  • “Denatured” describes the state of a peptide in solution that is characterized by a large conformational freedom of the peptide backbone. Most peptides and proteins adopt a denatured conformation in the presence of high concentrations of denaturants or at elevated temperature. Peptides in denatured conformation have, for example, characteristic circular dichroism (CD) spectra and are characterized by a lack of long-range interactions as determined by NMR.
  • CD characteristic circular dichroism
  • the invention provides XTEN sequences that, under physiologic conditions, resemble denatured sequences that are largely devoid in secondary structure. In other cases, the XTEN sequences are substantially devoid of secondary structure under physiologic conditions.
  • “Largely devoid,” as used in this context, means that less than 50% of the XTEN amino acid residues of the XTEN sequence contribute to secondary structure as measured or determined by the means described herein.
  • “Substantially devoid,” as used in this context, means that at least about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or at least about 99% of the XTEN amino acid residues of the XTEN sequence do not contribute to secondary structure, as measured or determined by the methods described herein.
  • Secondary structure can be measured spectrophotometrically, e.g., by circular dichroism spectroscopy in the "far-UV" spectral region (190-250 nm). Secondary structure elements, such as alpha-helix and beta-sheet, each give rise to a characteristic shape and magnitude of CD spectra. Secondary structure can also be predicted for a polypeptide sequence via certain computer programs or algorithms, such as the well-known Chou-Fasman algorithm (Chou, P. Y., et al.
  • the XTEN sequences used in the subject fusion protein compositions can have an alpha-helix percentage ranging from 0% to less than about 5% as determined by the Chou- Fasman algorithm. In other cases, the XTEN sequences of the fusion protein compositions have a beta- sheet percentage ranging from 0% to less than about 5% as determined by the Chou-Fasman algorithm. In some embodiments, the XTEN sequences of the fusion protein compositions have an alpha-helix percentage ranging from 0% to less than about 5% and a beta-sheet percentage ranging from 0% to less than about 5% as determined by the Chou-Fasman algorithm.
  • the XTEN sequences of the fusion protein compositions have an alpha-helix percentage less than about 2% and a beta-sheet percentage less than about 2%. In other cases, the XTEN sequences of the fusion protein compositions have a high degree of random coil percentage, as determined by the GOR algorithm.
  • an XTEN sequence have at least about 80%, more preferably at least about 90%, more preferably at least about 91%, more preferably at least about 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, and most preferably at least about 99% random coil, as determined by the GOR algorithm.
  • XTEN sequences of the compositions are substantially non-repetitive.
  • repetitive amino acid sequences have a tendency to aggregate or form higher order structures, as exemplified by natural repetitive sequences such as collagens and leucine zippers. These repetitive amino acids may also tend to form contacts resulting in crystalline or pseudocrystaline structures.
  • the low tendency of non-repetitive sequences to aggregate enables the design of long-sequence XTENs with a relatively low frequency of charged amino acids that would otherwise be likely to aggregate if the sequences were repetitive.
  • the CFXTEN fusion proteins comprise XTEN sequences of greater than about 100 to about 3000 amino acid residues wherein the sequences are substantially non-repetitive.
  • the XTEN sequences have greater than about 100 to about 3000 amino acid residues in which no three contiguous amino acids in the sequence are identical amino acid types unless the amino acid is serine, in which case no more than three contiguous amino acids are serine residues.
  • the XTEN sequence is "substantially non- repetitive.”
  • the degree of repetitiveness of a polypeptide or a gene can be measured by computer programs or algorithms or by other means known in the art. Repetitiveness in a polypeptide sequence can, for example, be assessed by determining the number of times shorter sequences of a given length occur WSGR Dkt Ref. 32808-726601 within the polypeptide. For example, a polypeptide of 200 amino acid residues has 192 overlapping 9- amino acid sequences (or 9-mer "frames") and 198 3-mer frames, but the number of unique 9-mer or 3- mer sequences will depend on the amount of repetitiveness within the sequence.
  • subsequence score A score is generated (hereinafter “subsequence score") that is reflective of the degree of repetitiveness of the subsequences in the overall polypeptide sequence.
  • “subsequence score” means the sum of occurrences of each unique 3-mer frame across a 200 consecutive amino acid sequence of the polypeptide divided by the absolute number of unique 3-mer subsequences within the 200 amino acid sequence. Examples of such subsequence scores derived from the first 200 amino acids of repetitive and non-repetitive polypeptides are presented in Example 44.
  • the present invention provides CFXTEN each comprising one or more XTEN in which the XTEN has a subsequence score less than 12, more preferably less than 10, more preferably less than 9, more preferably less than 8, more preferably less than 7, more preferably less than 6, and most preferably less than 5.
  • an XTEN with a subsequence score less than about 10 is “substantially non-repetitive.”
  • the non-repetitive characteristic of XTEN imparts a CF fusion proteins a greater degree of solubility and less tendency to aggregate compared to polypeptides having repetitive sequences. These properties facilitate the formulation of XTEN-comprising pharmaceutical preparations containing extremely high drug concentrations, in some cases exceeding 100 mg ml.
  • the XTEN polypeptide sequences of the embodiments are designed to have a low degree of internal repetitiveness in order to reduce or substantially eliminate immunogenicity when administered to a mammal.
  • Polypeptide sequences composed of short, repeated motifs largely limited to three amino acids, such as glycine, serine and glutamate, may result in relatively high antibody titers when administered to a mammal despite the absence of predicted T-cell epitopes in these sequences.
  • the present invention encompasses XTEN used as fusion partners that comprise multiple units of shorter sequences, or motifs, in which the amino acid sequences of the motifs are non-repetitive.
  • the non-repetitive criterion can be met despite the use of a "building block” approach using a library of sequence motifs that are multimerized to create the XTEN sequences.
  • an XTEN sequence may consist of multiple units of as few as four different types of sequence motifs, because the motifs themselves generally consist of non-repetitive amino acid sequences, the overall XTEN sequence is rendered substantially non-repetitive.
  • XTEN have a non-repetitive sequence of greater than about 100 to about 3000 amino acid residues wherein at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97%, or about 100% of the XTEN sequence consists of non-overlapping sequence motifs, wherein each of the motifs has about 9 to 36 amino acid residues.
  • At least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97%, or about 100% of the XTEN sequence consists of non-overlapping sequence motifs wherein each of the motifs has 9 to 14 amino acid residues.
  • at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97%, or about 100% of the XTEN sequence component consists of non-overlapping sequence motifs wherein each of the motifs has 12 amino acid residues.
  • the sequence motifs be composed mainly of small hydrophilic amino acids, such that the overall sequence has an unstructured, flexible characteristic.
  • amino acids that are included in XTEN are, e.g., arginine, lysine, threonine, alanine, asparagine, glutamine, aspartate, glutamate, serine, and glycine.
  • XTEN compositions with enhanced characteristics mainly include glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues wherein the sequences are designed to be substantially non-repetitive.
  • XTEN sequences have predominately four to six types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) or proline (P) that are arranged in a substantially non-repetitive sequence that is greater than about 100 to about 3000 amino acid residues, preferably greater than 400 to about 3000 residues in length.
  • G glycine
  • A alanine
  • S serine
  • T threonine
  • E glutamate
  • P proline
  • XTEN have sequences of greater than about 100 to about 3000 amino acid residues wherein at least about 80% of the sequence consists of non-overlapping sequence motifs wherein each of the motifs has 9 to 36 amino acid residues wherein each of the motifs consists of 4 to 6 types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), and wherein the content of any one amino acid type in the full-length XTEN does not exceed 30%.
  • G glycine
  • A alanine
  • S serine
  • T threonine
  • P proline
  • At least about 90% of the XTEN sequence consists of non-overlapping sequence motifs wherein each of the motifs has 9 to 36 amino acid residues wherein the motifs consist of 4 to 6 types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), and wherein the content of any one amino acid type in the full-length XTEN does not exceed 30%.
  • G glycine
  • A alanine
  • S serine
  • T threonine
  • P proline
  • At least about 90% of the XTEN sequence consists of non-overlapping sequence motifs wherein each of the motifs has 12 amino acid residues consisting of 4 to 6 types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), and wherein the content of any one amino acid type in the full-length XTEN does not exceed 30%.
  • At least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, to about 100% of the XTEN sequence consists of non-overlapping sequence motifs wherein each of the motifs has 12 amino acid WSGR Dkt.
  • Ref. 32808-726601 residues consisting of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), and wherein the content of any one amino acid type in the full-length XTEN does not exceed 30%.
  • XTENs comprise non-repetitive sequences of greater than about 100 to about 3000 amino acid residues wherein at least about 80%, or at least about 90%, or about 91 %, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% of the sequence consists of non-overlapping sequence motifs of 9 to 14 amino acid residues wherein the motifs consist of 4 to 6 types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), and wherein the sequence of any two contiguous amino acid residues in any one motif is not repeated more than twice in the sequence motif.
  • G glycine
  • A alanine
  • S serine
  • T threonine
  • P proline
  • At least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% of an XTEN sequence consists of non- overlapping sequence motifs of 12 amino acid residues wherein the motifs consist of 4 to 6 types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), and wherein the sequence of any two contiguous amino acid residues in any one sequence motif is not repeated more than twice in the sequence motif.
  • At least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% of an XTEN sequence consists of non-overlapping sequence motifs of 12 amino acid residues wherein the motifs consist of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), and wherein the sequence of any two contiguous amino acid residues in any one sequence motif is not repeated more than twice in the sequence motif.
  • XTENs consist of 12 amino acid sequence motifs wherein the amino acids are selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), and wherein the sequence of any two contiguous amino acid residues in any one sequence motif is not repeated more than twice in the sequence motif, and wherein the content of any one amino acid type in the full-length XTEN does not exceed 30%.
  • G glycine
  • A alanine
  • S serine
  • T threonine
  • P proline
  • the invention provides compositions comprising non-repetitive XTEN sequence(s) of greater than about 100 to about 3000 amino acid residues wherein at least about 80%, or at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% to about 100% of the sequence consists of multiple units of two or more non-overlapping sequence motifs selected from the amino acid sequences of Table 3.
  • the XTEN comprises non-overlapping sequence motifs in which about 80%, or at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% to about 100% of the sequence consists of two or more non- overlapping sequences selected from a single motif family of Table 3, resulting in a "family" sequence in which the overall sequence remains substantially non-repetitive. Accordingly, in these embodiments, an XTEN sequence comprises multiple units of non-overlapping sequence motifs of the AD motif family, or WSGR Dkt. Ref.
  • the XTEN comprises motif sequences from two or more of the motif families of Table 3.
  • the CFXTEN composition comprises a non-repetitive XTEN sequence of greater than about 100 to about 3000 amino acid residues, wherein at least about 80%, or at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% to about 100% of the sequence consists of non-overlapping 36 amino acid sequence motifs selected from one or more of the polypeptide sequences of Tables 9-12.
  • the XTEN component of the CFXTEN fusion protein has less than 100% of its amino acids consisting of four to six amino acid selected from glycine (G), alanine (A), WSGR Dkt Ref.
  • the other amino acid residues are selected from any other of the 14 natural L-amino acids, but are preferentially selected from hydrophilic amino acids such that the XTEN sequence contains at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% hydrophilic amino acids.
  • the XTEN amino acids that are not glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) are interspersed throughout the XTEN sequence, are located within or between the sequence motifs, or are concentrated in one or more short stretches of the XTEN sequence.
  • the XTEN component of the CFXTEN comprises amino acids other than glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P)
  • it is preferred that the amino acids not be hydrophobic residues and should not substantially confer secondary structure of the XTEN component.
  • Hydrophobic residues that are less favored in construction of XTEN include tryptophan, phenylalanine, tyrosine, leucine, isoleucine, valine, and methionine. Additionally, one can design the XTEN sequences to contain few (e.g. less than 5%) or none of the following amino acids: cysteine (to avoid disulfide formation and oxidation), methionine (to avoid oxidation), asparagine and glutamine (to avoid desamidation).
  • the XTEN component of the CFXTEN fusion protein comprising other amino acids in addition to glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) would have a sequence with less than 5% of the residues contributing to alpha-helices and beta-sheets as measured by the Chou-Fasman algorithm and have at least 90%, or at least about 95% or more random coil formation as measured by the GOR algorithm.
  • the invention encompasses CFXTEN compositions comprising carriers of XTEN polypeptides with extended length sequences.
  • the present invention makes use of the discovery that increasing the length of the non-repetitive, unstructured polypeptides enhances the unstructured nature of the XTENs and correspondingly enhances the biological and pharmacokinetic properties of fusion proteins comprising the XTEN carrier.
  • proportional increases in the length of the XTEN even if created by a fixed repeat order of single family sequence motifs (e.g., the four AE motifs of Table 3), result in a sequence with a higher percentage of random coil formation, as determined by GOR algorithm, compared to shorter XTEN lengths.
  • increasing the length of the unstructured polypeptide fusion partner results in a fusion protein with a disproportionate increase in terminal half-life compared to fusion proteins with unstructured polypeptide partners with shorter sequence lengths.
  • Non-limiting examples of XTEN contemplated for inclusion in the CFXTEN of the invention are presented in Table 4, below.
  • the invention provides CFXTEN compositions wherein the XTEN sequence length of the fusion protein(s) is greater than about 100 to about 3000 amino acid residues, and in some cases is greater than 400 to about 3000 amino acid residues, wherein the WSGR Dkt. Ref. 32808-726601
  • the XTEN sequences of the CFXTEN compositions of the present invention can be about 100, or about 144, or about 288, or about 401 , or about 500, or about 600, or about 700, or about 800, or about 900, or about 1000, or about 1500, or about 2000, or about 2500 or up to about 3000 amino acid residues in length.
  • the XTEN sequences can be about 100 to 150, about 150 to 250, about 250 to 400, 401 to about 500, about 500 to 900, about 900 to 1500, about 1500 to 2000, or about 2000 to about 3000 amino acid residues in length.
  • the CFXTEN can comprise an XTEN sequence wherein the sequence exhibits at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to a XTEN selected from Table 4.
  • the XTEN sequence is designed for optimized expression as the N-terminal component of the CFXTEN by inclusion of encoding nucleotides for an optimized N-terminal leader sequence (NTS) in the XTEN portion of the gene encoding the fusion protein.
  • NTS N-terminal leader sequence
  • the N-terminal XTEN sequence of the expressed CFXTEN has at least 90% sequence identity compared to the sequence of AE48 or AM48, AE624, or AE912 or AM923. In another embodiment, the XTEN has the N-terminal residues described in Examples 14-17.
  • the CFXTEN fusion protein comprises a first and a second XTEN sequence, wherein the cumulative total of the residues in the XTEN sequences is greater than about 400 to about 3000 amino acid residues and the XTEN can be identical or they can be different in sequence.
  • the CFXTEN fusion protein comprises a first and a second XTEN sequence wherein the sequences each exhibit at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to at least a first or additionally a second XTEN selected from Table 4.
  • Examples where more than one XTEN is used in a CFXTEN composition include, but are not limited to constructs with an XTEN linked to both the N- and C-termini of at least one CF.
  • the invention provides methods in which the CFXTEN is designed by selecting the length of the XTEN to confer a target half-life on a fusion protein administered to a subject.
  • XTEN lengths longer that about cumulative 400 residues incorporated into the CFXTEN compositions result in longer half-life compared to shorter cumulative lengths; e.g., shorter than about 280 residues.
  • CFXTEN fusion proteins are designed to comprise XTEN with a longer sequence length that is selected to additionally confer slower rates of systemic absorption after subcutaneous or intramuscular administration to a subject. In such
  • the C max is reduced in comparison to a comparable dose of a CF not linked to XTEN, thereby contributing to the ability to keep the CFXTEN within the therapeutic window for the composition.
  • the XTEN confers the property of a depot to the administered CFXTEN, in addition to the other physical/chemical properties described herein.
  • the invention provides an isolated CFXTEN fusion protein wherein the cumulative length of the XTEN component is greater than about 100 to about 3000 amino acid residues containing at least one polypeptide sequence segment selected from Tables 4, 9, 10, 1 1, 12, and 13 and wherein at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98% or more of the remainder of the XTEN sequence contains hydrophilic amino acids and less than about 2% of the remainder of the XTEN consists of hydrophobic or aromatic amino acids or cysteine.
  • the XTEN contains multiple segments wherein the segments are identical or different.
  • the invention provides an isolated CFXTEN fusion protein wherein the cumulative length of the XTEN component is greater than about 100 to about 3000 amino acid residues and comprises at least one sequence segment of at least about 100 to about 923, or at least about 100 to about 875, or at least about 100 to about 576, or at least about 100 to about 288, or at least about 100 to about 144 amino acid residues wherein the sequence segment(s) consists of at least three different types of amino acids and the sum of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues in the sequence segment(s) constitutes at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or
  • the invention provides an isolated CFXTEN fusion protein wherein the cumulative length of the XTEN component is greater than about 100 to about 3000 amino acid residues and comprises at least one sequence segment of at least about 200 to about 923, or at least about 200 to about 875, or at least about 200 to about 576, or at least about 200 to about 288 amino acid residues wherein the sequence segment(s) the sum of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues in the sequence segment(s) constitutes at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% of the total amino acid sequence of the sequence segment and wherein the subsequence score of the segment is less than 12, more preferably less than 10, more preferably less
  • the invention provides a short-length XTEN sequence incorporated as the N-terminal portion of the CFXTEN fusion protein. It has been discovered that the expression of the fusion protein is enhanced in a host cell transformed with a suitable expression vector comprising an optimized N-terminal leader polynucleotide sequence (that encodes the N-terminal XTEN) incorporated into the polynucleotide encoding the binding fusion protein.
  • a host cell transformed with such an expression vector comprising an optimized N-terminal leader sequence (NTS) in the binding fusion protein gene results in greatly-enhanced expression of the fusion protein compared to the expression of a corresponding fusion protein from a polynucleotide not comprising the NTS, and obviates the need for incorporation of a non-XTEN leader sequence used to enhance expression.
  • NTS N-terminal leader sequence
  • the invention provides CFXTEN fusion proteins comprising an NTS wherein the expression of the binding fusion protein from the encoding gene in a host cell is enhanced about 50%, or about 75%, or about 100%, or about 150%, or about 200%, or about 400% compared to expression of a CFXTEN fusion protein not comprising the N-terminal XTEN sequence (where the encoding gene lacks the NTS).
  • the N-terminal XTEN polypeptide of the CFXTEN comprises a sequence that exhibits at least about 80%, more preferably at least about 90%, more preferably at least about 91%, more preferably at least about 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, more preferably at least 99%, or exhibits 100% sequence identity compared to the amino acid sequence of AE48 or AM48, the respective amino acid sequences of which are as follows:
  • AE48 MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGS
  • AM48 MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGS
  • the short-length N-terminal XTEN is linked to an XTEN of longer length to form the N-terminal region of the CFXTEN fusion protein, wherein the polynucleotide sequence encoding the short-length N-terminal XTEN confers the property of enhanced expression in the host cell, and wherein the long length of the expressed XTEN contributes to the enhanced properties of the XTEN carrier in the fusion protein, as described above.
  • the short-length XTEN is linked to any of the XTEN disclosed herein (e.g., an XTEN of Table 3) and the resulting XTEN, in turn, is linked to the N-terminal of any of the CF disclosed herein (e.g., a CF of Table 1 or Table 2) as a component of the fusion protein.
  • polynucleotides encoding the short-length XTEN (or its complement) is linked to polynucleotides encoding any of the XTEN (or its complement) disclosed herein and the resulting gene encoding the N-terminal XTEN, in turn, is linked to the 5' end of polynucleotides encoding any of the CF (or to the 3 ' end of its complement) disclosed herein.
  • the N- terminal XTEN polypeptide with long length exhibits at least about 80%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least 99%, or exhibits 100% sequence WSGR Dkt.
  • the N-terminal XTEN can have from about one to about six additional amino acid residues, preferably selected from GESTPA, to accommodate the restriction endonuclease restriction sites that is employed to join the nucleotides encoding the N-terminal XTEN to the gene encoding the targeting moiety of the fusion protein.
  • additional amino acid residues preferably selected from GESTPA
  • the XTEN polypeptides have an unstructured characteristic imparted by incorporation of amino acid residues with a net charge and/or reducing the proportion of hydrophobic amino acids in the XTEN sequence.
  • the overall net charge and net charge density is controlled by modifying the content of charged amino acids in the XTEN sequences.
  • the net charge density of the XTEN of the compositions may be above +0.1 or below -0.1 charges/residue.
  • net charge density of a protien or peptide herein is meant the net charge divided by the total number of amino acids in the protein or proptide.
  • the net charge density of a XTEN can be about 0%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10% about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% or more.
  • the XTEN sequences are designed to have a net negative charge to minimize non-specific interactions between the XTEN containing compositions and various surfaces such as blood vessels, healthy tissues, or various receptors.
  • the XTEN can adopt open conformations due to electrostatic repulsion between individual amino acids of the XTEN polypeptide that individually carry a net negative charge and that are distributed across the sequence of the XTEN polypeptide.
  • Such a distribution of net negative charge in the extended sequence lengths of XTEN can lead to an unstructured conformation that, in turn, can result in an effective increase in hydrodynamic radius.
  • the negative charge is conferred by incorporation of glutamic acid residues.
  • the invention provides XTEN in which the XTEN sequences contain about 8, 10, 15, 20, 25, or even about 30% glutamic acid. Generally, the glutamic residues is spaced uniformly across the XTEN sequence. In some cases, the XTEN can contain about 10-80, or about 15-60, or about 20-50 glutamic residues per 20kDa of XTEN that can result in an XTEN with charged residues that would have very similar pKa, which can increase the charge homogeneity of the product and sharpen its isoelectric point, enhance the physicochemical properties of the resulting CFXTEN fusion protein for, and hence, simplifying purification procedures.
  • the XTEN of the compositions of the present invention generally have no or a low content of positively charged amino acids.
  • the XTEN may have less than about 10% amino WSGR Dkt. Ref. 32808-726601 acid residues with a positive charge, or less than about 7%, or less than about 5%, or less than about 2%, or less than about 1% amino acid residues with a positive charge.
  • the invention contemplates constructs where a limited number of amino acids with a positive charge, such as lysine, are incorporated into XTEN to permit conjugation between the epsilon amine of the lysine and a reactive group on a peptide, a linker bridge, or a reactive group on a drug or small molecule to be conjugated to the XTEN backbone.
  • a limited number of amino acids with a positive charge such as lysine
  • the XTEN has between about 1 to about 100 lysine residues, or about 1 to about 70 lysine residues, or about 1 to about 50 lysine residues, or about 1 to about 30 lysine residues, or about 1 to about 20 lysine residues, or about 1 to about 10 lysine residues, or about 1 to about 5 lysine residues, or alternatively only a single lysine residue.
  • fusion proteins are constructed that comprises XTEN, a coagulation factor, plus a chemotherapeutic agent useful in the treatment of growth-related diseases or disorders, wherein the maximum number of molecules of the agent incorporated into the XTEN component is determined by the numbers of lysines or other amino acids with reactive side chains (e.g., cysteine) incorporated into the XTEN.
  • the XTEN sequence comprises charged residues separated by other residues such as serine or glycine, which leads to better expression or purification behavior. Based on the net charge, some XTENs have an isoelectric point (pi) of 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, or even 6.5. In preferred embodiments, the XTEN will have an isoelectric point between 1.5 and 4.5. In these embodiments, the XTEN incorporated into the CFXTEN fusion protein compositions of the present invention carry a net negative charge under physiologic conditions that contribute to the unstructured conformation and reduced binding of the XTEN component to mammalian proteins and tissues.
  • the invention provides that the content of hydrophobic amino acids in the XTEN will typically be less than 5%, or less than 2%, or less than 1% hydrophobic amino acid content.
  • the amino acid content of methionine and tryptophan in the XTEN component of a CFXTEN fusion protein is typically less than 5%, or less than 2%, and most preferably less than 1%.
  • the XTEN will have a sequence that has less than 10% amino acid residues with a positive charge, or less than about 7%, or less that about 5%, or less than about 2% amino acid residues with a positive charge, the sum of methionine and tryptophan residues will be less than 2%, and the sum of asparagine and glutamine residues will be less than 10% of the total XTEN sequence.
  • the invention provides compositions in which the XTEN sequences have a low degree of immunogenicity or are substantially non-immunogenic.
  • Several factors can contribute to the low immunogenicity of XTEN, e.g., the non-repetitive sequence, the unstructured conformation, the high degree of solubility, the low degree or lack of self-aggregation, the low degree or lack of proteolytic sites within the sequence, and the low degree or lack of epitopes in the XTEN sequence.
  • the non-repetitive sequence e.g., the non-repetitive sequence, the unstructured conformation, the high degree of solubility, the low degree or lack of self-aggregation, the low degree or lack of proteolytic sites within the sequence, and the low degree or lack of epitopes in the XTEN sequence.
  • the non-repetitive sequence e.g., the unstructured conformation, the high degree of solubility, the low degree or lack of self-aggregation, the low degree or lack of prote
  • Conformational epitopes are formed by regions of the protein surface that are composed of multiple discontinuous amino acid sequences of the protein antigen.
  • the precise folding of the protein brings these sequences into a well-defined, stable spatial configurations, or epitopes, that can be recognized as "foreign" by the host humoral immune system, resulting in the production of antibodies to the protein or the activation of a cell-mediated immune response.
  • the immune response to a protein in an individual is heavily influenced by T-cell epitope recognition that is a function of the peptide binding specificity of that individual's HLA-DR allotype.
  • T-cell receptor on the surface of the T-cell, together with the cross-binding of certain other co-receptors such as the CD4 molecule, can induce an activated state within the T-cell. Activation leads to the release of cytokines further activating other lymphocytes such as B cells to produce antibodies or activating T killer cells as a full cellular immune response.
  • a peptide to bind a given MHC Class II molecule for presentation on the surface of an APC is dependent on a number of factors; most notably its primary sequence.
  • a lower degree of immunogenicity is achieved by designing XTEN sequences that resist antigen processing in antigen presenting cells, and/or choosing sequences that do not bind MHC receptors well.
  • the invention provides CFXTEN fusion proteins with substantially non- repetitive XTEN polypeptides designed to reduce binding with MHC II receptors, as well as avoiding formation of epitopes for T-cell receptor or antibody binding, resulting in a low degree of
  • avoidance of immunogenicity can attribute to, at least in part, a result of the conformational flexibility of XTEN sequences; i.e., the lack of secondary structure due to the selection and order of amino acid residues.
  • sequences having a low tendency to adapt compactly folded conformations in aqueous solution or under physiologic conditions that could result in conformational epitopes.
  • the administration of fusion proteins comprising XTEN using conventional therapeutic practices and dosing, would generally not result in the formation of neutralizing antibodies to the XTEN sequence, and also reduce the immunogenicity of the CF fusion partner in the CFXTEN compositions.
  • the XTEN sequences utilized in the subject fusion proteins can be substantially free of epitopes recognized by human T cells.
  • the elimination of such epitopes for the purpose of generating less immunogenic proteins has been disclosed previously; see for example WO 98/52976, WO 02/079232, and WO 00/3317 which are incorporated by reference herein.
  • Assays for human T cell epitopes have been described (Stickler, M., et al. (2003) J Immunol Methods, 281: 95-108).
  • peptide sequences that can be oligomerized without generating T cell epitopes or non-human sequences.
  • the XTEN sequences are substantially non-immunogenic by the restriction of the numbers of epitopes of the XTEN predicted to bind MHC receptors. With a reduction in the numbers of epitopes SGR Dkt. Ref. 32808-726601 capable of binding to MHC receptors, there is a concomitant reduction in the potential for T cell activation as well as T cell helper function, reduced B cell activation or upregulation and reduced antibody production.
  • the low degree of predicted T-cell epitopes can be determined by epitope prediction algorithms such as, e.g., TEPITOPE (Sturniolo, T., et al. (1999) Nat Biotechnol, 17: 555-61), as shown in Example 45.
  • the TEPITOPE score of a given peptide frame within a protein is the log of the K d (dissociation constant, affinity, off-rate) of the binding of that peptide frame to multiple of the most common human MHC alleles, as disclosed in Sturniolo, T. et al. (1999) Nature Biotechnology 17:555).
  • an XTEN component incorporated into a CFXTEN does not have a predicted T-cell epitope at a TEPITOPE score of about -5 or greater, or -6 or greater, or -7 or greater, or -8 or greater, or at a TEPITOPE score of -9 or greater.
  • a score of "-9 or greater” would encompass TEPITOPE scores of 10 to -9, inclusive, but would not encompass a score of -10, as -10 is less than -9.
  • the inventive XTEN sequences are rendered substantially non-immunogenic by the restriction of known proteolytic sites from the sequence of the XTEN, reducing the processing of XTEN into small peptides that can bind to MHC II receptors.
  • the XTEN sequence is rendered substantially non-immunogenic by the use a sequence that is substantially devoid of secondary structure, conferring resistance to many proteases due to the high entropy of the structure.
  • an XTEN of a CFXTEN fusion protein can have > 100 nM K ⁇ j binding to a mammalian receptor, or greater than 500 nM Kj, or greater than 1 ⁇ 3 ⁇ 4 towards a mammalian cell surface or circulating polypeptide receptor.
  • the non-repetitive sequence and corresponding lack of epitopes of XTEN limit the ability of B cells to bind to or be activated by XTEN.
  • a repetitive sequence is recognized and can form multivalent contacts with even a few B cells and, as a consequence of the cross-linking of multiple T-cell independent receptors, can stimulate B cell proliferation and antibody production.
  • each individual B cell may only make one or a small number of contacts with an individual XTEN due to the lack of repetitiveness of the sequence.
  • XTENs typically have a much lower tendency to stimulate proliferation of B cells and thus an immune response.
  • the CFXTEN have reduced immunogenic ity as compared to the corresponding CF that is not fused to an XTENT.
  • the administration of up to three parenteral doses of a CFXTEN to a mammal result in detectable anti-CFXTEN IgG at a serum dilution of 1 : 100 but not at a dilution of 1 : 1000.
  • the administration of up to three parenteral doses of a CFXTEN to a WSGR Dkt Ref. 32808-726601 mammal result in detectable anti-CF IgG at a serum dilution of 1 : 100 but not at a dilution of 1 : 1000.
  • the administration of up to three parenteral doses of a CFXTEN to a mammal result in detectable anti-XTEN IgG at a serum dilution of 1 : 100 but not at a dilution of 1 : 1000.
  • the mammal can be a mouse, a rat, a rabbit, or a cynomolgus monkey.
  • Non-repetitive XTENs form weaker contacts with antibodies.
  • Antibodies are multivalent molecules. For instance, IgGs have two identical binding sites and IgMs contain 10 identical binding sites. Thus antibodies against repetitive sequences can form multivalent contacts with such repetitive sequences with high avidity, which can affect the potency and/or elimination of such repetitive sequences.
  • antibodies against non-repetitive XTENs may yield monovalent interactions, resulting in less likelihood of immune clearance such that the CFXTEN compositions can remain in circulation for an increased period of time.
  • the present invention provides XTEN in which the XTEN polypeptides have a high hydrodynamic radius that confers a corresponding increased apparent molecular weight to the CFXTEN fusion protein incorporating the XTEN.
  • the linking of XTEN to CF sequences, such as FD or FVII sequences results in CFXTEN compositions that can have increased hydrodynamic radii, increased apparent molecular weight, and increased apparent molecular weight factor compared to a CF not linked to an XTEN.
  • compositions in which a XTEN with a high hydrodynamic radius is incorporated into a fusion protein comprising CF can effectively enlarge the hydrodynamic radius of the composition beyond the glomerular pore size of approximately 3-5 nra (corresponding to an apparent molecular weight of about 70 kDA, which is larger than both native FIX and FVII) (Caliceti. 2003.
  • the hydrodynamic radius of a protein is determined by its molecular weight as well as by its structure, including shape or compactness.
  • the XTEN can adopt open conformations due to electrostatic repulsion between individual charges of the peptide or the inherent flexibility imparted by the particular amino acids in the sequence that lack potential to confer secondary structure.
  • the open, extended and unstructured conformation of the XTEN polypeptide can have a greater proportional hydrodynamic radius compared to polypeptides of a comparable sequence length and/or molecular weight that have secondary and/or tertiary structure, such as typical globular proteins.
  • Methods for determining the hydrodynamic radius are well known in the art, such as by the use of size exclusion chromatography (SEC), as described in U.S. Patent Nos. 6,406,632 and 7,294,513.
  • the CFXTEN fusion protein can be configured with an XTEN such that the fusion protein can have a hydrodynamic radius of at least about 5 nm, or at least about 8 nm, or at least about 10 nm, or 12 nm, or at least about 15 nm.
  • the large hydrodynamic radius conferred by the XTEN in an CFXTEN fusion protein can lead to reduced renal clearance of the resulting fusion protein, leading to a corresponding increase in terminal half-life, an increase in mean residence time, and/or a decrease in renal clearance rate.
  • an XTEN of a chosen length and sequence can be selectively incorporated into a CFXTEN to create a fusion protein that have, under physiologic conditions, an apparent molecular weight of at least about 500 kDa, or at least about 800 kDa, or at least about 1000 kDa, or at least about 1500 kDA, or at least about 1800 kDa, or at least about 2000 kDa, or at least about 2300 kDa or more.
  • an XTEN of a chosen length and sequence can be selectively linked to a CF to result in a CFXTEN fusion protein that has, under physiologic conditions, an apparent molecular weight factor of at least four, alternatively of at least five, alternatively of at least six, alternatively of at least eight, alternatively of at least 10, alternatively of at least 15, or an apparent molecular weight factor of at least 20 or greater.
  • the CFXTEN fusion protein has, under physiologic conditions, an apparent molecular weight factor that is about 4 to about 20, or is about 6 to about 15, or is about 8 to about 12, or is about 9 to about 10 relative to the actual molecular weight of the fusion protein.
  • the CF of the subject compositions are not limited to native, full-length FIX or FVII polypeptides, but also include recombinant versions as well as biologically and/or pharmacologically active forms with sequence variants, combinations of FVII and FIX sequences, or fragments thereof.
  • recombinant versions as well as biologically and/or pharmacologically active forms with sequence variants, combinations of FVII and FIX sequences, or fragments thereof.
  • various amino acid deletions, insertions and substitutions can be made in the CF to create variants without departing from the spirit of the invention with respect to the biological activity or pharmacologic properties of the CF. Examples of conservative substitutions for amino acids in polypeptide sequences are shown in Table 5.
  • the invention contemplates substitution of any of the other 19 natural L-amino acids for a given amino acid residue of the given CF (e.g., FIX or FVII), which may be at any position within the sequence of the CF, including adjacent amino acid residues. If any one substitution results in an undesirable change in biological activity, then one of the alternative amino acids can be employed and the construct evaluated by the methods described herein, or using any of the techniques and guidelines for conservative and non-conservative mutations set forth, for instance, in U.S. Pat. No.
  • variants can include, for instance, polypeptides wherein one or more amino acid residues are added or deleted at the N- or C-terminus of the full-length native amino acid sequence of a CF that retains some if WSGR Dkt. Ref. 32808-726601 not all of the biological activity of the native peptide; e.g., the ability to activate another coagulation factor and/or participate in the coagulation cascade, leading to fibrin formation and hemostasis.
  • a factor EX incorporated into a CFXTEN fusion protein has a sequence that exhibits at least about 80% sequence identity compared to a sequence from Table 1 , alternatively at least about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, sequence identity as compared with a sequence from Table 1.
  • a factor VII incorporated into a CFXTEN fusion protein has a sequence that exhibits at least about 80% sequence identity compared to a sequence from Table 2, alternatively at least about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, sequence identity as compared with a sequence from Table 2.
  • the present invention encompasses CFXTEN that comprise one or more XTEN sequences located internal to the CF sequence.
  • the one or more internally-located XTEN can be a sequence length of 36 to >1000 amino acid residues.
  • the CFXTEN can have one or two or three or four or more XTEN sequences with at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or WSGR Dkt. Ref. 32808-726601
  • the CFXTEN with one or more internal XTEN has an additional XTEN located at the N- or C- terminus of the fusion protein with at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to one or more XTEN selected from Table 4.
  • the invention provided CFXTEN with internal XTEN (as detailed below) that further comprises a C-terminus XTEN linked to the CF by a cleavage sequence (e.g., a cleavage sequence of Table 7) such that the XTEN can be released when acted on by a protease.
  • a cleavage sequence e.g., a cleavage sequence of Table 7.
  • an XTEN can be located between the domains of a FIX sequence; e.g., between the Gla and EGF1, or between the EGF 1 and EGF2, or between the EGF2 and the activation peptide, or within the sequence of the activation peptide between the R145-A146 and R180-V181 activation peptide residues of the AP (i.e., between any two amino acids of the sequence TVFPDVDYVNSTEAETILDNITQSTQSFNDF), or between the EGF2 and the activation peptide, or between the activation peptide and the protease domain, or any combination of the foregoing.
  • a FIX sequence e.g., between the Gla and EGF1, or between the EGF 1 and EGF2, or between the EGF2 and the activation peptide, or within the sequence of the activation peptide between the R145-A146 and R180-V181 activation peptide residues of the AP (i.e., between any two
  • the XTEN can be inserted within an existing loop sequence within an individual domain of the FIX sequence so that 1) the XTEN forms a looped structure outside the domain and doesn't disrupt the normal architecture of the domain; and 2) the XTEN can be released by cleavage of incorporated cleavage sites.
  • the invention provides a CFXTEN comprising a FVII that incorporates one or more XTEN located between the domains of a FVII sequence; e.g., between the Gla and EGF1, or between the EGF 1 and EGF2, or between the EGF2 and the activating peptide, or between the activating peptide and the protease domain, or any combination of the foregoing.
  • the XTEN can be a sequence of 36 to >1000 amino acid residues including, but not limited to a sequence that has at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% or more sequence identity compared to a sequence from Table 4, 8, 9, 10, 11, 12, and 13. In one embodiment, as illustrated in FIGS.
  • an XTEN is incorporated between the EGF2 domain and the single lytic cleavage site at residues Arg 152 -Ile 1 53 .
  • the XTEN can be inserted within an existing loop sequence within an individual domain of the FVII sequence so that 1) the XTEN forms a looped structure outside the domain and doesn't disrupt the normal architecture of the domain; and 2) the XTEN can be released by cleavage of incorporated cleavage sites.
  • the invention provides an isolated factor VII polypeptide comprising at least one heterologous sequence that is cleavable by a pro-coagulant protease that does not activate a wildtype factor VII, wherein upon cleavage heterologous sequence, the factor VII polypeptide is activated.
  • a pro-coagulant protease that does not activate a wildtype factor VII, wherein upon cleavage heterologous sequence, the factor VII polypeptide is activated.
  • CFXTEN with factor Vll-factor IX hybrid sequence variants that incorporate into, or replace a portion of the sequence, a factor VII construct portions of the activating peptide domain (AP) sequence from factor ⁇ , resulting in hybrid compositions that can be activated as part of the intrinsic system of the coagulation cascade.
  • the CFXTEN that incorporate such factor Vll-factor DC sequence variants as the CF component of the fusion protein permit administration to a subject a composition in which the CF component is not activated, and can be dosed at high amounts because it remains as an inert, circulating depot that is largely resistant to inactivation by protease inhibitors until activated by the triggering of the intrinsic coagulation cascade or by auto-activation, the latter a slow process.
  • FVII FDC hybrid sequences are illustrated in FIG. 36, showing those portions of the hybrid amino acid sequences that have homology with those of native FIX and FVH
  • the CFXTEN comprise factor Vll-factor DC sequence variants that substitute portions or the entirety of the F C activating peptide sequence with one or both FDC AP cleavage sites for FVII sequence to the N-terminal side of the protease domain of FVII; i.e., either towards the N-terminus beginning with the arginine at position 212 of the full- length precursor polypeptide or the isoleucine at position 213.
  • the factor Vll-factor DC sequence CF incorporates the full-length FDC AP domain plus at least about 2, or at least about 3, or at least about 4, or at least about 5, or at least about 6, or at least about 7, or at least about 8, or at least about 9, or at least about 10, or at least about 11, or at least about 12 amino acids flanking adjacent amino acid residues on one or both sides of the R145-A146 and R180-V181 cleavage sites of FDC (e.g., the sequence RVSVSQTS LTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRWGGE in the case of 12 flanking amino acids on the N-terminus side and 5 flanking amino acids on the C-terminus side).
  • FDC e.g., the sequence RVSVSQTS LTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRWGGE in the case of 12 flanking amino acids on the N-terminus side and 5 flanking amino acids on the C-terminus side.
  • the CFXTEN comprises a factor Vll-factor DC sequence variant that incorporates a portion of the AP that includes a sequence of at least about 2, or at least about 3, or at least about 4, or at least about 5 that flank the R145-A146 AP cleavage site (e.g., the sequence TSKLTRAETVFP in the case of 6 flanking amino acids on either side of the cleavage site).
  • a factor Vll-factor DC sequence variant that incorporates a portion of the AP that includes a sequence of at least about 2, or at least about 3, or at least about 4, or at least about 5 that flank the R145-A146 AP cleavage site (e.g., the sequence TSKLTRAETVFP in the case of 6 flanking amino acids on either side of the cleavage site).
  • the CFXTEN comprises a factor Vll-factor DC sequence variant that incorporates a portion of the AP that includes a sequence of at least about 2, or at least about 3, or at least about 4, or at least about 5 amino acids that flank one or both sides of the Rl 80-V181 AP cleavage site (e.g., the sequence and DFTRV in the case of 4 amino acids on the N-terminal flank and valine as the C-terminus of the cleavage site from FDC).
  • the sequence and DFTRV in the case of 4 amino acids on the N-terminal flank and valine as the C-terminus of the cleavage site from FDC.
  • the CFXTEN comprises the factor Vll-factor DC sequence variant of any of the foregoing embodiments of this paragraph that further includes the same AP sequence as a linker between the C-terminus of the FVII component and the XTEN component of the fusion protein; e.g., an N- to C- terminus configuration of FVII variant- AP sequence-XTEN, thereby permitting the release of the factor Vll-factor DC sequence variant component from the CFXTEN fusion protein by the same intrinsic coagulation factor as per that of the FVII to FVIIa transition.
  • the CFXTEN comprises the factor Vll-factor DC sequence variant of any of the foregoing embodiments of this paragraph that further includes the factor XI cleavage sequence KLTRAET as the linker between the FVII variant sequence and the XTEN, thereby permitting the release of the factor Vll-factor DC sequence WSGR Dkt Ref. 32808-726601 variant component from the CFXTEN fusion protein by the initiation of the intrinsic coagulation cascade.
  • the activated factor Vll-factor DC sequence variant would have a shorter half-life compared to the intact CFXTEN, thereby increasing the margin of safety and tolerability of the composition in a subject.
  • the activated factor Vll-factor DC sequence variant molecule can have at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95% of the biological activity as native FVIIa, as measured by any of the appropriate assays or parameters disclosed herein (e.g., PT or bleeding time assays).
  • the invention provides the factor VH-factor DC sequence variants of the foregoing embodiments of this paragraph without a linked XTEN, permitting their administration to a subject as a circulating depot of the factor Vll-factor IX hybrid that can be activated by either the intrinsic or extrinsic coagulation cascade.
  • the invention provides a CFXTEN with a factor Vll-factor DC sequence variant with incorporated FDC-derived sequence with an overall sequence that exhibits at least about 80% sequence identity, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, sequence identity compared to a sequence from Table 43.
  • the invention provides a factor Vll-factor DC sequence variant with incorporated FDC-derived cleavage sequence (without an XTEN) with a sequence that exhibits at least about 80% sequence identity, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, sequence identity as compared with a sequence from Table 43 without an XTEN.
  • the CFXTEN comprising factor Vll-factor DC sequence variants can be evaluated for biological activity using assays or in vivo parameters as described herein (e.g., in vitro coagulation assays or a pharmacodynamic effect in a hemophilia model), and those sequences that retain at least about 40%, or about 50%, or about 55%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95% or more activity compared to the corresponding native FVII sequence is considered suitable for inclusion in the subject CFXTEN.
  • the CF found to retain a suitable level of activity can be linked to one or more XTEN polypeptides described hereinabove.
  • a CF found to retain a suitable level of activity can be linked to one or more XTEN polypeptides having at least about 80% sequence identity to a sequence from Table 4, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% sequence identity as compared with a sequence of Table 4, resulting in a chimeric fusion protein.
  • the invention provides CFXTEN fusion protein compositions with the CF and XTEN components linked in specific N- to C-terminus configurations.
  • one or more CFs are linked to one or more XTENs, either at the N-terminus or at the C-terminus, with or without a spacer, to form a block copolymer, and the sequential arrangement of the CFs and the XTENs in the CFXTEN fusion protein are the same as the configuration known in the block copolymer chemistry. When there is Ref.
  • each of the CF, the XTEN, or the spacer have the same or different sequences, and the CFs and/or XTENs are linked either continuously or alternately (regular or irregular).
  • the CFXTEN is a monomeric fusion protein with a CF linked to one XTEN polypeptide.
  • the CFXTEN is a monomeric fusion protein with a CF linked to two or more XTEN polypeptides.
  • the CFXTEN is a monomeric fusion protein with two or more CF linked to one XTEN polypeptide. In still other embodiments, the CFXTEN is a monomeric fusion protein with two or more CF linked to two or more XTEN polypeptide. In still other embodiment, the CFXTEN is a monomeric fusion protein with a single CF in which XTEN is located within the CF sequence (e.g., within a FIX sequence such as between one or more domains as illustrated in FIGS. 2 and 5).
  • Table 6 provides non- limiting examples of configurations that are encompassed by the CFXTEN fusion proteins of the invention; numerous other variations will be apparent to the ordinarily skilled artisan, including the incorporation the spacer and cleavage sequences disclosed herein or known in the art.
  • the invention contemplates CFXTEN fusion proteins compositions comprising, but not limited to single or multiple CF selected from Table 1 or Table 2 (or fragments or sequence variants thereof ⁇ single or multiple XTEN selected from Table 4 (or sequence variants thereof) that are in a configuration shown in Table 6.
  • Non-limiting examples of sequences of fusion proteins containing a single CF linked to a single XTEN are presented in Table 41.
  • a CFXTEN composition would comprise a fusion protein having at least about 80% sequence identity compared to a CFXTEN from Table 41, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% sequence identity as compared to a CFXTEN from Table 41.
  • the resulting CFXTEN retains at least a portion of the biological activity of the corresponding CF not linked to the XTEN.
  • the CFXTEN fusion protein can further comprise a cleavage sequence from Table 7; the cleavage sequence being located between the CF and the XTEN or between adjacent CF (if more than one CF is included in the CFXTEN).
  • the CFXTEN comprising the cleavage sequences will also have one or more spacer sequence amino acids between the CF and the cleavage sequence or the XTEN and the cleavage sequence to facilitate access of the protease; the spacer amino acids comprising any natural amino acid, including glycine and alanine as preferred amino acids.
  • Non- limiting examples of CFXTEN comprising CF, XTEN, cleavage sequence(s) and spacer amino acids are presented in Table 42.
  • the invention also contemplates substitution of any of the CF sequences of Tables 1 and 2 for a CF sequence of Table 42, substitution of any XTEN sequence of Table 4 for an XTEN sequence of Table 42, and substitution of any cleavage sequence of Table 7 for a cleavage sequence of Table 42.
  • the CF component either becomes biologically active or has an increase in activity upon its release from the XTEN by cleavage of the cleavage sequence(s), described more fully below.
  • the invention provides a fusion protein of formula I:
  • CF is a coagulation factor
  • x is either 0 or 1
  • y is either 0 or 1 wherein x+y >1
  • XTEN is an extended recombinant polypeptide
  • the invention provides a fusion protein of formula ⁇ :
  • CF is a coagulation factor a
  • S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence
  • x is either 0 or 1
  • y is either 0 or 1 wherein x+y >1
  • XTEN is an extended recombinant polypeptide.
  • the invention provides an isolated fusion protein, wherein the fusion protein is of formula III:
  • CF is a coagulation factor
  • S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence
  • x is either 0 or 1 and y is either 0 or 1 wherein x+y >1
  • XTEN is an extended recombinant polypeptide.
  • the invention provides an isolated fusion protein of formula IV:
  • Gla is a Gla domain of FIX
  • EGF1 is an EGF1 domain of FIX
  • EGF2 is an EFG2 domain of FIX
  • AP is an activator peptide of FIX
  • PRO is a protease domain of FIX
  • S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence
  • u is either 0 or 1
  • v is either 0 or 1
  • x is either 0 or 1
  • y is either 0 or 1
  • z is either 0 or 1, with the proviso that u + v + x + z >l
  • XTEN is an extended recombinant polypeptide.
  • the invention provides an isolated fusion protein of formula V:
  • Gla is a Gla domain of FDC
  • EGF1 is an EGF1 domain of FDC
  • EGF2 is an EFG2 domain of FDC
  • API is the N-terminal sequence portion of the activator peptide domain of FDC that includes a first native cleavage sequence of the AP domain
  • AP2 is the C-terminal sequence portion of the activator peptide domain of FDC that includes a second native cleavage sequence of the AP domain
  • PRO is a protease domain of FDC
  • S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence
  • u is either 0 or 1
  • v is either 0 or 1
  • w is 0 or 1
  • x is either 0 or 1
  • y is either 0 or 1
  • z is either 0 or 1 with the proviso that u + v + w+ x + z >l
  • XTEN is an extended recombinant
  • the invention provides an isolated fusion protein of formula VI:
  • Gla is a Gla domain of FVII
  • EGF1 is an EGF1 domain of FVII
  • EGF2 is an EFG2 domain of FVII
  • PRO is a protease domain of FVII
  • S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence
  • u is either 0 or 1
  • v is either 0 or 1
  • x is either 0 or 1
  • y is either 0 or 1
  • XTEN is an extended recombinant polypeptide.
  • the invention provides an isolated fusion protein of formula VII: WSGR Dkt. Ref. 32808-726601
  • Gla is a Gla domain of FVII
  • EGF1 is an EGF1 domain of FVII
  • EGF2 is an EFG2 domain of FVII
  • PRO is a protease domain of FVII
  • API is the N-terminal sequence portion of the activator peptide domain of FIX that includes the native cleavage sequence
  • AP2 is the C-terminal sequence portion of the activator peptide domain of FIX that includes the native cleavage sequence
  • S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence
  • t is either 0 or 1
  • u is either 0 or 1
  • v is either 0 or 1
  • the factor VII variant includes can include one or both cleavage sequences from the activator peptide domain of factor IX; e.g., a sequence of at least about 2, or at least about 3, or at least about 4, or at least about 5 amino acids that flank the R145-A146 cleavage site (e.g., the sequence TSKLTRAETVFP in the case of 5 flanking amino acids) and the sequence of at least about 2, or at least about 3, or at least about 4, or at least about 5 amino acids that flank the Rl 80-V 181 cleavage site (e.g., the sequence FNDFTRWGGED in the case of 5 flanking amino acids, as described more fully above.
  • the invention also contemplates substitution of any of the other cleavage sequences of Table 7 for the AP sequences of the factor VII variant.
  • the embodiments of formulae V and VI encompass CFXTEN configurations of factor IX and factor VII, respectively, wherein one or more XTEN of lengths ranging from about 36 amino acids to > 1000 amino acids (e.g., sequences selected from Tables 4, and 9-13) are inserted and linked between adjoining domains of the factor EX or the factor VII sequence, respectively.
  • the invention contemplates all possible permutations of insertions of XTEN between the domains of either FIX or FVII with optional linking of an additional XTEN to the C-terminus of the FIX or the FVII, optionally via an additional cleavage sequence selected from Table 7, resulting in a CFXTEN composition; non-limiting examples of which are portrayed in FIGS. 2, 5 and 6.
  • the CFXTEN fusion proteins can be evaluated for retention of biological activity (including after cleavage of any incorporated XTEN-releasing cleavage sites) using any appropriate in vitro assay disclosed herein (e.g., the assays of Table 40 or the assays described in the Examples), to determine the suitability of the configuration for use as a therapeutic agent in the treatment of a coagulation-factor related disease, disorder or condition.
  • any appropriate in vitro assay disclosed herein e.g., the assays of Table 40 or the assays described in the Examples
  • administration of a therapeutically effective amount of a fusion protein of one of formulae I-VII to a subject in need thereof results in an increase of at least two-fold in the terminal half-life, or at least three-fold, or at least four-fold, or at least five-fold, or at least 10-fold, or at least 20-fold, or at least 40-fold, or at least 100-fold increase in the terminal half-life for the fusion protein compared to the corresponding CF not linked to the XTEN and administered at a comparable amount administered to a subject.
  • administration of a therapeutically effective amount of a fusion protein of one of formulae I-VII to a subject in need thereof results in a gain in time of at least two-fold, or at least three-fold, or at least four-fold, or at least five-fold, or at least 10-fold, or at least 20- WSGR Dkt.
  • administration of a therapeutically effective dose of a fusion protein of one of formulae I-VII to a subject in need thereof can result in a gain in time between consecutive doses necessary to maintain a therapeutically effective blood level of the fusion protein of at least 48 h, or at least 72 h, or at least about 96 h, or at least about 120 h, or at least about 7 days, or at least about 14 days, or at least about 21 days between consecutive doses compared to a CF not linked to XTEN and administered at a comparable dose.
  • any spacer sequence group optionally is intorduced to a subject fusion protein encompassed by the invention.
  • the spacer is provided to enhance expression of the fusion protein from a host cell or to decrease steric hindrance such that the CF component may assume its desired tertiary structure and/or interact appropriately with its target substrate.
  • the spacer comprises one or more peptide sequences that are between 1-50 amino acid residues in length, or about 1-25 residues, or about 1-10 residues in length.
  • Spacer sequences can comprise any of the 20 natural L amino acids, and will preferably comprise hydrophilic amino acids that are sterically unhindered that can include, but not be limited to, glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P).
  • the spacer can be polyglycines or polyalanines, or is predominately a mixture of combinations of glycine and alanine residues.
  • spacer polypeptide exclusive of a cleavage sequence is largely to substantially devoid of secondary structure; e.g., less than about 10%, or less than about 5% as determined by the Chou-Fasman and/or GOR algorithms.
  • a spacer sequence in a CFXTEN fusion protein composition further contains one or more cleavage sequences, which are identical or different, wherein the cleavage sequence may be acted on by a protease to release the CF from the fusion protein.
  • the incorporation of the cleavage sequence into the CFXTEN is designed to permit release of a CF that becomes active or more active upon its release from the XTEN; e.g., the enzymatic activity of the CF component increases.
  • the CF that becomes active after release is a FIX or a sequence variant thereof.
  • the CF that becomes active after release is a FVII or a sequence variant thereof.
  • the cleavage sequences are located sufficiently close to the CF sequences, generally within 18, or within 12, or within 6, or within 2 amino acids of the CF sequence terminus, such that any remaining residues attached to the CF after cleavage do not appreciably interfere with the activity (e.g., such as binding to a ligand or substrate) of the CF, yet provide sufficient access to the protease to be able to effect cleavage of the cleavage sequence.
  • the cleavage site is a sequence that can be cleaved by a protease endogenous to the mammalian subject such that the CFXTEN can be cleaved after
  • the CFXTEN can serve as a prodrug or a circulating depot for WSGR Dkt. Ref. 32808-726601 the CF.
  • the CF that is released from the fusion protein by cleavage of the cleavage sequence exhibits at least about a two-fold, or at least about a three-fold, or at least about a four-fold, or at least about a five-fold, or at least about a six-fold, or at least about a eight-fold, or at least about a tenfold, or at least about a 20-fold increase in enzymatic activity for its native substrate compared to the intact CFXTEN fusion protein.
  • cleavage sites contemplated by the invention include, but are not limited to, a polypeptide sequence cleavable by a mammalian endogenous protease selected from FXIa, FXIIa, kallikrein, FVIIa, FEXa, FXa, Flla (thrombin), Elastase-2, granzyme B, MMP-12, MMP-13, MMP-17 or MMP-20, or by non-mammalian proteases such as TEV, enterokinase, PreScissionTM protease (rhinovirus 3C protease), and sortase A.
  • a mammalian endogenous protease selected from FXIa, FXIIa, kallikrein, FVIIa, FEXa, FXa, Flla (thrombin), Elastase-2, granzyme B, MMP-12, MMP-13, MMP-17 or MMP-20, or by non-ma
  • Sequences known to be cleaved by the foregoing proteases and others are known in the art. Exemplary cleavage sequences and cut sites within the sequences are presented in Table 7, as well as sequence variants thereof.
  • thrombin activate clotting factor ⁇
  • LTPRSLLV sequence LTPRSLLV
  • Active Flla is produced by cleavage of FII by FXa in the presence of phospholipids and calcium and is down stream from factor ⁇ in the coagulation pathway.
  • the invention provides CFXTEN comprising one or more cleavage sequences operably positioned to release the CF from the fusion protein upon cleavage, wherein the one or more cleavage sequences has at least about 86%, or at least about 92% or greater sequence identity to a sequence selected from Table 7.
  • the CFXTEN comprising a cleavage sequence would have at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity compared to a sequence selected from Table 42.
  • the known cleavage sequence have one or more deletions or insertions or one or two or three amino acid substitutions for any one or two or three amino acids in the known sequence, wherein the deletions, insertions or substitutions result in reduced or WSGR Dkt Ref. 32808-726601 enhanced susceptibility but not an absence of susceptibility to the protease, resulting in an ability to tailor the rate of release of the CF from the XTEN. Exemplary substitutions are shown in Table 7.
  • the invention provides CFXTEN fusion proteins with enhanced pharmacokinetics compared to the CF not linked to XTEN.
  • the pharmacokinetic properties of a CF that can be enhanced by linking a given XTEN to the CF include, but are not limited to, terminal half-life, area under the curve (AUC), Cm a , volume of distribution, and bioavailability; properties that provide enhanced utility in the treatment of coagulation factor-related disorders, diseases and related conditions.
  • the CFXTEN when used at the dose and dose regimen determined to be appropriate for the composition by the methods described herein, can achieve a circulating concentration resulting in a desired pharmacologic effect, yet stay within the safety range for biologically active component of the composition for an extended period of time compared to a comparable dose of the CF not linked to XTEN.
  • the CFXTEN remains within the therapeutic window for the fusion protein composition for the extended period of time compared to a CF not liked to XTEN and administered to a WSGR Dkt. Ref. 32808-726601 subject at a comparable dose.
  • a “comparable dose” means a dose with an equivalent moles/kg for the active CF pharmacophore (e.g., FIX or FVII) that is administered to a subject in a comparable fashion. It will be understood in the art that a "comparable dosage" of CFXTEN fusion protein would represent a greater weight of agent but would have essentially the same mole-equivalents of CF in the dose of the fusion protein administered.
  • the CFXTEN with enhanced pharmacokinetic properties can be a sequence that has at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to a protein sequence selected from any one of Tables 41, 42, or 43.
  • the CFXTEN with enhanced pharmacokinetic properties can comprise a CF sequence that has at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% sequence identity compared to a sequence from Table 1 or from Table 2, linked to one or more XTEN that has at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% sequence identity compared to a sequence from Table 4.
  • CFXTEN with a longer terminal half-life are generally preferred, so as to improve patient convenience, to increase the interval between doses and to reduce the amount of drug required to achieve a sustained effect.
  • the administration of the fusion protein results in an improvement in at least one of the parameters (disclosed herein as being useful for assessing the subject diseases, conditions or disorders) using a lower unit dose in moles of fusion protein compared to the corresponding CF component not linked to the fusion protein and administered at a comparable unit dose or dose regimen to a subject.
  • the total dose in moles administered to achieve the improvement is at least about three-fold lower, or at least about fourfold, or at least about five-fold, or at least about six-fold, or at least about eight-fold, or at least about 10- fold lower compared to the corresponding CF component not linked to the fusion protein.
  • the invention provides CFXTEN fusion proteins comprising XTEN wherein the XTEN is selected to provide a targeted half-life for the CFXTEN composition administered to a subject.
  • the invention provides monomeric fusion proteins comprising XTEN wherein the XTEN is selected to confer an increase in the terminal half-life for the CFXTEN administered to a subject, compared to the corresponding CF not linked to the fusion protein and administered at a comparable dose, wherein the increase is at least about two-fold longer, or at least about three-fold, or at least about fourfold, or at least about five-fold, or at least about six-fold, or at least about seven-fold, or at least about eight-fold, or at least about nine-fold, or at least about ten-fold, or at least about 15-fold, or at least a 20- fold, or at least a 40-fold, or at least a 80-fold, or at least a 100-fold or greater an increase in terminal half- WSGR Dkt.
  • Exogenously administered factor IX has been reported to have a terminal half-life in humans of approximately 18-24 hours (Morfini, M. Blood Transfus. (2008) 6(s2): s21-s25) and exogenously administered factor VII is reported to have a terminal half-life of approximately 4-6 hours (Klitgaard T, Br J Clin Pharmacol (2008) 65(1 ):3-1 1), whereas various CFXTEN compositions disclosed herein that have been experimentally administered to animals, as described in the Examples, have resulted in terminal half-life values considerably longer.
  • the present invention provides CFXTEN fusion proteins that exhibits an increase in ACU of at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about a 100%, or at least about 150%, or at least about 200%, or at least about 300%, or at least about 500%, or at least about 1000%, or at least about a 2000% compared to the corresponding CF not linked to the XTEN and administered to a subject at a comparable dose.
  • the pharmacokinetic parameters of a CFXTEN can be determined by standard methods involving dosing, the taking of blood samples at times intervals, and the assaying of the protein using ELISA, HPLC, radioassay, or other methods known in the art or as described herein, followed by standard calculations of the data to derive the half-life and other PK parameters.
  • the enhanced PK parameters allow for reduced dosing of the CFXTEN compositions, compared to CF not linked to XTEN.
  • a smaller molar amount of about two-fold less, or about three-fold less, or about four-fold less, or about five-fold less, or about six-fold less, or about eight-fold less, or about 10-fold less or greater of the fusion protein is administered in comparison to the corresponding CF not linked to the XTEN under a dose regimen needed to maintain hemostasis, and the fusion protein achieves a comparable area under the curve as the corresponding molar amount of the CF not linked to the XTEN.
  • the fusion protein has a less frequent administration regimen of about every two days, about every seven days, about every 14 days, about every 21 days, or about monthly of the fusion protein administered to a subject, compared to the daily administration of an otherwise same dose amount of the corresponding CF not linked to the XTEN, and the fusion protein achieves a comparable area under the curve as the corresponding CF not linked to the XTEN.
  • an accumulative smaller molar amount of about 5%, or about 10%, or about 20%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90% less of the fusion protein is administered to a subject in comparison to the corresponding molar amount of the CF not linked to the XTEN under a dose regimen needed to maintain hemostasis, yet the fusion protein achieves at least a comparable area under the curve as the corresponding CF not linked to the XTEN.
  • the accumulative smaller molar amount is measure for a period of at least about one week, or about 14 days, or about 21 days, or about one month.
  • the invention further provides CFXTEN comprising a CF molecule separated from the XTEN sequence by one or more cleavage sequences; e.g., a sequence from Table 7.
  • the intact CFXTEN composition has less activity but a longer half-life in its intact form compared to a corresponding CF not linked to the XTEN, but is designed such that upon administration to a subject, the WSGR Dkt Ref. 32808-726601
  • the CF component is gradually released from the fusion protein by cleavage at the cleavage sequence(s) by endogenous proteases, whereupon the CF component exhibits activity, i.e., the ability to effectively bind to and activate its target coagulation protein substrate.
  • the CFXTEN with a cleavage sequence has about 80% sequence identity compared to a sequence from Table 42, or about 85%, or about 90%, or about 95%, or about 97%, or about 98%, or about 99% sequence identity compared to a sequence from Table 42.
  • the CFXTEN of the foregoing embodiments in this paragraph serve as prodrugs or a circulating depot, resulting in a longer terminal half-life compared to CF not linked to the XTEN.
  • a higher concentration of CFXTEN can be administered to a subject, compared to the corresponding CF not linked to XTEN because a smaller proportion of the circulating composition is active.
  • the present invention provides CFXTEN compositions comprising CF covalently linked to XTEN that can have enhanced properties compared to CF not linked to XTEN, as well as methods to enhance the therapeutic and or biologic activity or effect of the respective two CF components of the compositions.
  • the invention provides CFXTEN compositions with enhanced properties compared to those art-known fusion proteins containing albumin, immunoglobulin polypeptide partners, polypeptides of shorter length and/or polypeptide partners with repetitive sequences.
  • CFXTEN fusion proteins provide significant advantages over chemical conjugates, such as pegylated constructs, notably the fact that recombinant CFXTEN fusion proteins can be made in bacterial cell expression systems, which can reduce time and cost at both the research and development and manufacturing stages of a product, as well as result in a more homogeneous, defined product with less toxicity for both the product and metabolites of the CFXTEN compared to pegylated conjugates.
  • the CFXTEN possesses a number of advantages over therapeutics not comprising XTEN, including one or more of the following non-limiting exemplary enhanced properties: increased solubility, increased thermal stability, reduced immunogenicity, increased apparent molecular weight, reduced renal clearance, reduced proteolysis, reduced metabolism, enhanced therapeutic efficiency, a lower effective therapeutic dose, increased bioavailability, increased time between dosages capable of maintaining blood levels within the therapeutic window for the CF, a "tailored" rate of absorption when administered subcutaneously or intramuscularly, enhanced lyophilization stability, enhanced serum/plasma stability, increased terminal half-life, increased solubility in blood stream, decreased binding by neutralizing antibodies, decreased active clearance, reduced side effects, retention of substrate binding affinity, stability to degradation, stability to freeze-thaw, stability to proteases, stability to ubiquitination, ease of administration, compatibility with other pharmaceutical excipients or carriers, persistence in the subject, increased stability in storage (e.g., increased shelf-life), reduced toxicity in an organism or
  • XTEN as a fusion partner increases the solubility of the CF payload.
  • the length and/or the motif family composition of the XTEN sequences incorporated into the fusion protein may each be selected to confer a different degree of solubility and/or stability on the respective fusion proteins such that the overall pharmaceutical properties of the CFXTEN composition are enhanced.
  • the CFXTEN fusion proteins can be constructed and assayed, using methods described herein, to confirm the physicochemical properties and the XTEN adjusted, as needed, to result in the desired properties.
  • the XTEN sequence of the CFXTEN is selected such that the fusion protein has an aqueous solubility that is within at least about 25% greater compared to a CF not linked to the fusion protein, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 75%, or at least about 100%, or at least about 200%, or at least about 300%, or at least about 400%, or at least about 500%, or at least about 1000% greater than the corresponding CF not linked to the fusion protein.
  • the invention provides methods to produce and recover expressed CFXTEN from a host cell with enhanced solubility and ease of recovery compared to CF not linked to XTEN.
  • the method includes the steps of transforming a eukaryotic host cell with a polynucleotide encoding a CFXTEN with one or more XTEN components of cumulative sequence length greater than about 200, or greater than about 400, or greater than about 600, or greater than about 800 amino acid residues, expressing the CFXTEN fusion protein in the host cell, and recovering the expressed fusion protein in soluble form.
  • the XTEN of the CFXTEN fusion proteins can have at least about 80% sequence identity, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, to about 100% sequence identity compared to one or more XTEN selected from Table 4 and the CF can have at least about 80% sequence identity, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, or WSGR Dkt. Ref. 32808-726601
  • components can be in an N- to C-terminus configuration selected from formulas I- VII.
  • the invention provides CFXTEN compositions and methods to produce the compositions that can maintain the CF component within a therapeutic window for a greater period of time compared to comparable dosages of the corresponding CF not linked to XTEN.
  • a "comparable dosage" of CFXTEN fusion protein would represent a greater weight of agent but would have the same approximate mole-equivalents of CF in the dose of the fusion protein and/or would have the same approximate molar concentration relative to the CF.
  • the method to produce the compositions that can maintain the CF component within a therapeutic window includes the steps of selecting the XTEN appropriate for conjugation to a CF to provide the desired pharmacokinetic properties in view of a given dose and dose regimen, administration of the CFXTEN to subjects in need thereof, followed by assays to verify the pharmacokinetic properties, the activity of the CFXTEN fusion protein, and the safety of the administered composition.
  • CFXTEN provided herein allow for increased efficacy of the administered composition by maintaining the circulating concentrations of the CF within the therapeutic window for an enhanced period of time.
  • therapeutic window means that the amount of drug or biologic as a blood or plasma concentration range, which provides efficacy or a desired pharmacologic effect over time for the disease or condition without unacceptable toxicity, i.e., the range of the circulating blood concentrations between the minimal amount to achieve any positive therapeutic effect and the maximum amount which results in a response that is the response immediately before toxicity to the subject (at a higher dose or concentration). Additionally, therapeutic window generally encompasses an aspect of time; the maximum and minimum concentration that results in a desired pharmacologic effect over time that does not result in unacceptable toxicity or adverse events. A dosed composition that stays within the therapeutic window for the subject could also be said to be within the "safety range.”
  • the characteristics of CFXTEN compositions of the invention can be determined by any suitable screening assay known in the art for measuring the desired characteristic.
  • the invention provides methods to assay the CFXTEN fusion proteins of differing composition or configuration in order to provide CFXTEN with the desired degree of biologic and/or therapeutic activity, as well as safety profile.
  • Specific in vivo and ex vivo biological assays are used to assess the activity of each configured CFXTEN and/or CF component to be incorporated into CFXTEN, including but not limited to the assays of the Examples, those assays of Table 40, as well as the following assays or other such assays known in the art for assaying the properties and effects of CF.
  • Functional assays can be conducted that allow determination of coagulation activity, such as prothrombin (PT) and activated partial prothrombin (aPTT) assays (Belaaouaj AA et al., J. Biol. Chem. (2000) 275:27123-8; Diaz-Collier JA.
  • PT prothrombin
  • aPTT activated partial prothrombin
  • CFXTEN for the target substrate of the corresponding CF WSGR Dkt.
  • Ref. 32808-726601 can be assayed using binding or competitive binding assays, such as Biacore assays with chip-bound receptors or binding proteins or ELISA assays, as described in US Patent 5,534,617, assays described in the Examples herein, radio-receptor assays, or other assays known in the art.
  • the foregoing assays can also be used to assess CF sequence variants (assayed as single components or as CFXTEN fusion proteins) and can be compared to the native CF to determine whether they have the same degree of biologic activity as the native CF, or some fraction thereof such that they are suitable for inclusion in CFXTEN; e.g., at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% of the activity compared to the native CF.
  • Dose optimization is important for all drugs, especially for those with a narrow therapeutic window.
  • a standardized single dose of CF for all patients presenting with a diverse symptoms or abnormal clinical parameters may not always be effective.
  • a consideration of these factors is well within the purview of the ordinarily skilled clinician for the purpose of determining the therapeutically or pharmacologically effective amount of the CFXTEN, versus that amount that would result in unacceptable toxicity and place it outside of the safety range, or insufficient potency such that clinical improvement is not achieved.
  • the therapeutic window for CF in subjects of different ages or degree of disease have been established and are available in published literature or are stated on the drug label for approved products containing the CF.
  • the therapeutic window can be established for new compositions, including those CFXTEN of the disclosure.
  • the methods for establishing the therapeutic window for a given composition are known to those of skill in the art (see, e.g., Goodman & Gilman's The Pharmacological Basis of Therapeutics, 11 th Edition, McGraw-Hill (2005)).
  • the therapeutic window for a given subject or population of subjects can be determined for a given drug or biologic, or combinations of biologies or drugs.
  • the dose escalation studies can evaluate the activity of a CFXTEN through metabolic studies in a subject or group of subjects that monitor physiological or biochemical parameters, as known in the art or as described herein for one or more parameters associated with the metabolic disease or disorder, or clinical parameters associated with a beneficial outcome for the particular indication, together with observations and/or measured parameters to determine the no effect dose, adverse events, maximum tolerated dose and the like, together with measurement of
  • pharmacokinetic parameters that establish the determined or derived circulating blood levels.
  • the results can then be correlated with the dose administered and the blood concentrations of the therapeutic that are coincident with the foregoing determined parameters or effect levels.
  • a range of doses and blood concentrations can be correlated to the minimum effective dose as well as the maximum dose and blood concentration at which a desired effect occurs and above which toxicity occurs, thereby establishing the therapeutic window for the dosed therapeutic.
  • Blood concentrations of the fusion protein (or as measured by the CF component) above the maximum is considered outside the therapeutic window WSGR Dkt Ref. 32808-726601 or safety range.
  • a Cmj- blood level is established, below which the CFXTEN fusion protein would not have the desired pharmacologic effect, and a Cmax blood level is established that would represent the highest circulating concentration before reaching a concentration that would elicit unacceptable side effects, toxicity or adverse events, placing it outside the safety range for the CFXTEN.
  • concentrations established, the frequency of dosing and the dosage can be further refined by measurement of the C,, ⁇ and Cmm to provide the appropriate dose and dose frequency to keep the fusion protein(s) within the therapeutic window.
  • One of skill in the art can, by the means disclosed herein or by other methods known in the art, confirm that the administered CFXTEN remains in the therapeutic window for the desired interval or requires adjustment in dose or length or sequence of XTEN. Further, the determination of the appropriate dose and dose frequency to keep the CFXTEN within the therapeutic window establishes the therapeutically effective dose regimen; the schedule for administration of multiple consecutive doses using a therapeutically effective dose of the fusion protein to a subject in need thereof resulting in consecutive C max peaks and/or C,nj n troughs that remain within the therapeutic window and results in an improvement in at least one measured parameter relevant for the target disease, disorder or condition.
  • the CFXTEN administered at an appropriate dose to a subject results in blood concentrations of the CFXTEN fusion protein that remains within the therapeutic window for a period at least about twofold longer compared to the corresponding CF not linked to XTEN and administered at a comparable dose; alternatively at least about three-fold longer; alternatively at least about four-fold longer;
  • an "appropriate dose” means a dose of a drug or biologic that, when administered to a subject, would result in a desirable therapeutic or pharmacologic effect and/or a blood concentration within the therapeutic window.
  • the CFXTEN administered at a therapeutically effective dose regimen results in a gain in time of at least about three-fold longer; alternatively at least about four-fold longer; alternatively at least about five-fold longer; alternatively at least about six-fold longer; alternatively at least about seven-fold longer; alternatively at least about eight-fold longer; alternatively at least about nine-fold longer or at least about ten-fold longer between at least two consecutive C max peaks and/or C ⁇ troughs for blood levels of the fusion protein compared to the corresponding biologically active protein of the fusion protein not linked to the fusion protein and administered at a comparable dose regimen to a subject.
  • the CFXTEN administered at a therapeutically effective dose regimen results in a comparable improvement in one, or two, or three or more measured parameter using less frequent dosing or a lower total dosage in moles of the fusion protein of the pharmaceutical composition compared to the corresponding biologically active protein component(s) not linked to the fusion protein and administered to a subject using a therapeutically effective dose regimen for the CF.
  • the measured WSGR Dkt. Ref. 32808-726601 parameters include any of the clinical, biochemical, or physiological parameters disclosed herein, or others known in the art for assessing subjects with coagulation factor-related disorders.
  • the CFXTEN fusion proteins of the invention retain at least about 0.05%, or about 0.1%, or about 1%, or about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 98%, or about 99% percent of the biological activity of the corresponding CF not linked to the fusion protein with regard to an in vitro biologic activity or pharmacologic effect known or associated with the use of the native CF in the treatment and prevention of coagulation factor-related diseases, disorders, and conditions.
  • Non- limiting examples of parameters or physiologic effects that can be assayed to assess the retained activity of the CFXTEN fusion proteins include prothrombin time (PT), activated partial thromboplastin time (aPTT), bleeding time, whole blood clotting time (WBCT), and thrombelastography.
  • PT prothrombin time
  • aPTT activated partial thromboplastin time
  • WBCT whole blood clotting time
  • thrombelastography thrombelastography
  • the activity of the CF component is manifested by the intact CFXTEN fusion protein, while in other cases the activity of the CF component is primarily manifested upon cleavage and release of the CF from the fusion protein by action of a protease that acts on a cleavage sequence incorporated into the CFXTEN fusion protein, embodiments of which are disclosed above.
  • the CFXTEN is designed to reduce the binding affinity of the CF component for the coagulation substrate when linked to the XTEN but have restored or increased affinity when released from XTEN through the cleavage of cleavage sequence(s) incorporated into the CFXTEN sequence, as described more fully above.
  • the invention provides an isolated fusion protein comprising a FIX linked to XTEN by a cleavage sequence, wherein the fusion protein is substantially inactive prior to cleavage and wherein the FIX released from the fusion protein by proteolytic cleavage at the cleavage sequence has biological activity that is at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95% as active compared to native FIX not linked to XTEN.
  • the CFXTEN can be designed to reduce active clearance of the CFXTEN to increase the terminal half-life of CFXTEN administered to a subject, while still retaining biological activity.
  • the clearance mechanisms to remove CF from the circulation have yet to be fully elucidated. Uptake, elimination, and inactivation of CFs can occur in the circulatory system as well as in the extravascular space.
  • Coagulation factors are complex proteins that interact with a large number of other proteins, lipids, and receptors, and many of these interactions can contribute to the elimination of CFs from the circulation.
  • clearance mechanisms for FVII a heterogeneously glycosylated protein, may include clearance by the liver.
  • the invention provides CFXTEN that reduce clearance of the fusion protein by linking one or more XTEN to the CF component of the fusion protein, wherein the fusion protein has an increase in apparent molecular weight factor of at least about four-fold, or at least about five-fold, or at least about six-fold, or at least about seven-fold, or at least about eight-fold, or at least about ten-fold, or at least about twelve-fold, or at least about fifteen-fold, and wherein the terminal half-life of the CFXTEN when administered to a subject is increased at least about two-fold, or at least about four-fold, or at least about eight-fold, or at least about 10-fold, or at least about 20-fold, or at least about 30-fold, or at least about 40-fold, or at least about 50-fold, or at least about 60- fold, or at least about 70-fold, or at
  • the XTEN can be identical or they can be of a different sequence composition (and net charge) or length.
  • Non-limiting examples of the foregoing embodiment with two XTEN linked to a single FVII are illustrated in FIG.
  • Gla-EGFl-EGF2-AE144-Protease-AE864 or Gla-EGFl-AE288-EGF2-Protease-AE864 (wherein the AE XTEN components have approximately a 17% net charge due to incorporated glutamic acid), Gla-EGF 1 - EGF2-AG144-Protease-AG864 or Gla-EGF 1 -AG 144-EGF2-Protease-AE864 (wherein the AG XTEN components have approximately no net charge).
  • the XTEN of the CFXTEN compositions with the higher net charge are expected, as described above, to have less nonspecific interactions with various negatively-charged surfaces such as blood vessels, tissues, or various receptors, which would further contribute to reduced active clearance.
  • the XTEN of the CFXTEN compositions with a low (or no) net charge are expected to have a higher degree of interaction with surfaces that, while contributing to active clearance, can potentiate the activity of the associated coagulation factor, given the known contribution of cell (e.g., platelets) and vascular surfaces to the coagulation process and the intensity of activation of coagulation factors (Zhou, R., et al., Biomaterials (2005) 26(16):2965-2973; London, F., et al. Biochemistry (2000) 39(32):9850-98 8).
  • the invention provides CFXTEN in which the degree of potency, bioavailability, and half-life can be tailored by the selection and placement of the type and length of the XTEN in the CFXTEN compositions.
  • the invention contemplates compositions in which a CF from Table 1 or from Table 2 and XTEN from Table 4 are substituted for the respective components of the foregoing examples, and are produced, for example, in a configuration from Table 6 or from formulas I-VII such that the construct has reduced clearance compared to an alternative configuration of the respective components.
  • the foregoing method for increasing the terminal half-life provides configured CFXTEN that can result in an increase in the terminal half-life of at least about 30%, or about 50%, or about 75%, or about 100%, or about 150%, or about 200%, or about 300%, or about 400% or more compared to the half-life of a CFXTEN in a second configuration where active clearance is not reduced.
  • VVSGR Dkt. Ref. 32808-726601 further takes advantage of the fact that certain ligands wherein reduced binding to a clearance receptor, either as a result of a decreased on-rate or an increased off-rate, may be effected by the obstruction of either the N- or C-terminus and using that terminus as the linkage to another polypeptide of the composition, whether another molecule of a CF, an XTEN, or a spacer sequence results in the reduced binding.
  • the choice of the particular configuration of the CFXTEN fusion protein reduces the degree of binding to a clearance receptor such that a reduced rate of active clearance is achieved.
  • the CFXTEN is designed to retain sufficient biologic activity for the intact molecule.
  • the invention provides a CFXTEN configured such that the biologic activity of the CFXTEN is in the range of about 0.01%-40%, or about 0.01%-30%, or about 0.01%-20%, or about 0.01%-10 of the biological activity compared to the corresponding native coagulation factor.
  • the biological activity of the configured CFXTEN is thus reduced by at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 99%, or at least about 99.99% as compared to the biological activity of the corresponding native coagulation factor not linked to XTEN, determined under comparable conditions.
  • the biological activity of the configured CFXTEN for the target receptor is "substantially reduced” compared to a corresponding native CF not linked to XTEN.
  • the present invention provides compositions and methods to produce compositions with reduced biological activity but increased half-life by configuring the CFXTEN, examples of which are provided above, so as to be able to provide a desired in vivo biological response yet avoid active clearance mechanisms.
  • the increased half-life permits higher dosages and reduced frequency of dosing compared to CF not linked to XTEN or compared to CFXTEN configurations wherein the fusion protein is subject to coagulation factor clearance mechanisms.
  • the invention provides a method for achieving a beneficial effect in bleeding disorders and/or in a coagulation factor-related disease, disorder or condition mediated by FIX or FVII.
  • coagulation factor-related diseases, disorders or conditions is intended to include, but is not limited to bleeding disorders (e.g., defective platelet function, thrombocytopenia or von Willebrand's disease), coagulopathies (any disorder of blood coagulation, including coagulation factor deficiencies), hemophilia B (aka Christmas disease), factor EX-related bleeding disorders, factor VII deficiency, hemophilia A, vascular injury, uncontrolled bleeding in subjects not suffering from hemophilia, bleeding from trauma or surgery, bleeding due to anticoagulant therapy, and bleeding due to liver disease or conditions that can be ameliorated or corrected by administration of FIX or FVII to a subject.
  • the present invention addresses disadvantages and/or limitations of other methods of treatment using factor ⁇ or factor VII preparations that have a relatively short terminal half-life and/
  • the invention provides methods for treating a subject, such as a human, with a coagulation factor-related disease, disorder or condition comprising the step of administering to the subject a therapeutically- or prophylactically-effective amount of an CFXTEN wherein said
  • the CFXTEN comprises a FVII. In another embodiment of the foregoing, the CFXTEN comprises a FD .
  • the effective amount produces a beneficial effect in helping to treat (e.g., cure or reduce the severity) or prevent (e.g., reduce the likelihood of onset or severity) a coagulation factor-related disease, disorder or condition.
  • treating means administering a drug or a biologic (e.g., a CFXTEN) to achieve an improvement in an existing disease, disorder or condition or preventing the occurrence of a disease, disorder or condition (including prophylaxis).
  • a therapeutically- effective amount of a CFXTEN fusion protein can be that amount of composition that, when administered as a single or as repeated doses to a subject, leads to improvements in or amelioration of the underlying disease, disorder or condition, or improvements in signs or symptoms or physiologic parameters associated with the underlying disease, disorder or condition.
  • Hemostasis is regulated by multiple protein factors, and such proteins, as well as analogues thereof, have found utility in the treatment of coagulation factor-related diseases, disorders and conditions.
  • the use of commercially-available coagulation factors has met with less than . optimal success in the management of subjects afflicted with such diseases, disorders and conditions.
  • dose optimization and frequency of dosing is important for coagulation factors used in the treatment or prevention of bleeding episodes in coagulation factor-related diseases, disorders, or conditions, or uncontrolled bleeding in subjects not suffering from hemophilia.
  • coagulation factors have a short half-life necessitates frequent dosing in order to achieve clinical benefit, which results in difficulties in the management of such patients.
  • the invention provides methods of treatment comprising administering a CFXTEN
  • the method of treatment comprises administering a therapeutically-effective amount of an CFXTEN composition to a subject suffering from hemophilia A wherein the administration results in the improvement of one or more biochemical or physiological parameters or clinical endpoints associated with the condition.
  • the method of treatment comprises administering a therapeutically-effective amount of an CFXTEN composition to a subject suffering from hemophilia B wherein the administration results in the improvement of one or more biochemical or physiological parameters or clinical endpoints associated with the condition.
  • the method of treatment comprises administering a therapeutically-effective amount of an CFXTEN composition to a subject suffering from factor VII deficiency wherein said administration results in the improvement of WSGR Dkt Ref. 32808-726601 one or more biochemical or physiological parameters or clinical endpoints associated with the condition.
  • the method of treatment comprises administering a therapeutically-effective amount of an CFXTEN composition to a subject suffering from or at risk of developing uncontrolled bleeding wherein the administration results in the improvement of one or more biochemical or physiological parameters or clinical endpoints associated with the condition.
  • the embodiments of the disclosed method of treatments utilizing a CFXTEN comprising a FVII are compositions in which the FVII has been activated; i.e., FVIIa.
  • the invention also contemplates CFXTEN compositions in which the FVII has not been activated.
  • the invention provides a method of treatment for a subject with a coagulation factor-related disease, disorder or conditions comprising administration of a CFXTEN comprising a FVII variant (as described above) wherein the FVII is not activated but has one or more cleavage sequences that, when cleaved by an endogenous protease, converts the FVII component to the activated form.
  • the method utilizes a CFXTEN composition that has a terminal half-life of at least about 12 h, or at least about 24 h, or at least about 48 h, or at least about 48 h, or at least about 96 h, or at least about 144 h, or at least about 160 h. Accordingly, the method represents a means to treat subjects with certain forms of chronic coagulopathies with what is essentially a "prodrug" form of FVII.
  • administration of the CFXTEN to a subject results in an improvement in one or more of the biochemical, physiologic, or clinical parameters that is of greater magnitude than that of the corresponding CF component not linked to XTEN, determined using the same assay or based on a measured clinical parameter.
  • administration of the CFXTEN to a subject using a therapeutically effective dose regimen results in activity in one or more of the biochemical, physiologic, or clinical parameters that is of longer duration than the activity of the corresponding CF component not linked to XTEN, determined using that same assay or based on a measured clinical parameter.
  • the administration of a therapeutically effective amount of a CFXTEN comprising a FVII to a subject results in a reduction in prothrombin time at about 2-7 days after administration of at least about 5%, or about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or more in the subject compared to the prothrombin time in a subject at a comparable time after administration of a comparable amount of FVII not linked to XTEN.
  • the administration of a CFXTEN comprising a FVII to a subject using a therapeutically effective amount results in maintenance of prothrombin times within 30% of normal in the subject for a period of time that WSGR Dkt Ref. 32808-726601 is at least two-fold, or about three-fold, or at least about four-fold longer compared to a comparable dose regimen of FVII not linked to XTEN administered to a subject.
  • administration of a therapeutically effective amount of a CFXTEN comprising a FIX to a subject results in a reduction in the activated partial prothrombin time at about 2-7 days after administration of at least about 5%, or about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or more in the subject compared to the activated partial prothrombin time in a subject at a comparable time after administration of a comparable amount of FIX not linked to XTEN.
  • the administration of a CFXTEN comprising a FIX to a subject using a therapeutically effective amount results in maintenance of activated partial prothrombin times within 30% of normal in the subject for a period of time that is at least two-fold, or at least about three-fold, or at least about fourfold longer compared to a comparable dose regimen of FIX not linked to XTEN administered to a subject.
  • the administration of a CFXTEN comprising a FVII to a subject using a therapeutically effective amount results in maintenance of a bleeding time (in a bleeding time assay) within 30% of normal in the subject for a period of time that is at least two-fold, or about three-fold, or at least about four-fold longer compared to a comparable amount of FVII not linked to XTEN administered to a subject.
  • the administration of a CFXTEN comprising a FIX to a subject using a therapeutically effective amount results in maintenance of a bleeding time (in a bleeding time assay) within 30% of normal in the subject for a period of time that is at least two-fold, or about threefold, or at least about four-fold longer compared to a comparable amount of FIX not linked to XTEN administered to a subject.
  • CFXTEN As a result of the enhanced PK parameters of CFXTEN, as described herein, the CF is administered using longer intervals between doses compared to the corresponding CF not linked to XTEN to prevent, treat, alleviate, reverse or ameliorate symptoms or clinical abnormalities of the coagulation factor-related disease, disorder or condition or prolong the survival of the subject being treated.
  • CFXTEN comprising FVII have utility in the treatment of hemophilia A and hemophilia B.
  • FVIIa administered in high concentrations can function as a bypassing agent resulting in the activation of FX even in the absence of FIX or FVITI.
  • FVIIa In order to act as a bypassing agent FVIIa has to be dosed at concentrations that exceed the level of FVIIa in healthy people by approximately 100-fold. These levels are generally safe because FVIIa has low activity in the absence of tissue factor (TF), to which FVII binds. Tissue factor is released or presented on injured tissues which triggers clotting via the extrinsic system.
  • TF tissue factor
  • the circulation half-life of FVIIa is in part limited by its inactivation by antithrombin (AT). Antithrombin can not bind to FVII but only to FVIIa.
  • the invention provides a method of treating hemophilia A or B by administering an amount of CFXTEN comprising an activated form of FVII, wherein the ability to activate FX in the circulation of a subject is maintained for a period that is at least about two-fold longer, or at least about three-fold, or at least about four-fold, or at least about five-fold, or at least about 10-fold, or at least about 20-fold longer WSGR Dkt.
  • Ref. 32808-726601 compared to FVII not linked to XTEN and administered to a comparable subject at a comparable dose.
  • the current invention further provides CFXTEN fusion proteins comprising FVII linked to XTEN that can not be inactivated by AT by more than about 5% prior to its activation to FVIIa-XTEN.
  • the invention provides a method of treatment comprising administering a CFXTEN with a FVII component that is not activated, wherein the CFXTEN serves as a circulating depot wherein the area under the curve for the FVII that is activated to FVIIa and not complexed with AT is at least about twofold greater, or at least about three-fold, or at least about four-fold, or at least about five-fold, or at least about 10-fold, or at least about 20-fold greater than a FVII not linked to XTEN and administered at a comparable dose.
  • a smaller molar amount of e.g. of about two-fold less, or about three-fold less, or about four-fold less, or about five-fold less, or about six-fold less, or about eight-fold less, or about 10-fold-less or greater
  • the fusion protein is administered in comparison to the corresponding CF not linked to the XTEN under an otherwise same dose regimen, and the fusion protein achieves a comparable therapeutic effect as the corresponding CF not linked to the XTEN;
  • the fusion protein is administered less frequently (e.g., every two days, about every seven days, about every 14 days, about every 21 days, or about, monthly) in comparison to the corresponding CF not linked to the XTEN under an otherwise same dose amount, and the fusion protein achieves a comparable therapeutic effect as the corresponding CF not linked to the XTEN; or
  • an accumulative smaller molar amount e.g.
  • the fusion protein achieves a comparable therapeutic effect as the corresponding CF not linked to the XTEN.
  • the accumulative smaller molar amount is measure for a period of at least about one week, or about 14 days, or about 21 days, or about one month.
  • the therapeutic effect can be determined by any of the measured parameters or clinical endpoints described herein.
  • the methods of the invention includes administration of consecutive doses of a therapeutically effective amount of the CFXTEN for a period of time sufficient to achieve and/or maintain the desired parameter or clinical effect, and such consecutive doses of a therapeutically effective amount establishes the therapeutically effective dose regimen for the CFXTEN, i.e., the schedule for consecutively administered doses of the fusion protein composition, wherein the doses are given in therapeutically effective amounts to result in a sustained beneficial effect on any clinical sign or symptom, aspect, measured parameter or characteristic of a coagulation factor-related disease state or condition, including, but not limited to, those described herein.
  • the method comprises administering a therapeutically-effective amount of a pharmaceutical composition comprising a CFXTEN fusion protein composition comprising a CF linked to an XTEN sequence(s) and at least one pharmaceutically acceptable carrier to a subject in need thereof that results in greater improvement in at least one parameter, physiologic condition, or clinical outcome mediated by the CF component(s) (non-limiting WSGR Dkt Ref. 32808-726601 examples of which are described above) compared to the effect mediated by administration of a pharmaceutical composition comprising a CF not linked to XTEN and administered at a comparable dose.
  • the pharmaceutical composition is administered at a therapeutically effective dose.
  • the pharmaceutical composition is administered using multiple consecutive doses using a therapeutically effective dose regimen (as defined herein) for the length of the dosing period.
  • a therapeutically effective amount of the CFXTEN varies according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the administered fusion protein to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the CFXTEN are outweighed by the therapeutically beneficial effects.
  • a prophylactically effective amount refers to an amount of CFXTEN required for the period of time necessary to achieve the desired prophylactic result; e.g., delayed onset of a bleeding episode.
  • the dose of the CFXTEN that is administered to a subject ranges from about 0.5 mg to 1000 mg dose, or from about 1 mg to 400 mg dose, or from about 10 mg to about 300 mg/dose for a 70 kg subject as loading and maintenance doses, depending on the weight of the subject and the severity of the condition.
  • the method of treatment comprises administration of a CFXTEN using a therapeutically effective dose regimen to effect improvements in one or more parameters associated with coagulation factor diseases, disorders or conditions.
  • administration of the CFXTEN to a subject results in an improvement in one or more of the biochemical, physiologic, or clinical parameters that is of greater magnitude than that of the corresponding CF component not linked to XTEN, determined using the same assay or based on a measured clinical parameter.
  • administration of the CFXTEN to a subject using a therapeutically effective dose regimen results in activity in one or more of the biochemical, physiologic, or clinical parameters that is of longer duration than the activity of one of the single CF components not linked to XTEN, determined using that same assay or based on a measured clinical parameter.
  • the administration of the CFXTEN to a subject using a therapeutically effective dose regimen results in an improvement in prothrombin time or activated partial thromboplastin time of at least about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 100% or more in the subject compared to a comparable dose of CF not linked to XTEN administered to a subject.
  • the administration of the CFXTEN to a subject using a therapeutically effective dose regimen results in decreased instances of bleeding in the subject of at least about 10%, or about 20%, or about 30%, or about 40%, or about 50% or more compared to a comparable dose regimen of CF not linked to XTEN administered to a subject.
  • CFXTEN used in accordance with the methods provided herein is administered in conjunction with other treatment methods and compositions (e.g., other coagulation proteins) useful for treating coagulation factor-related diseases, disorders, and conditions, or WSGR Dkt. Ref. 32808-726601 conditions for which coagulation factor is adjunctive therapy; e.g., bleeding episodes due to injury or surgery.
  • other treatment methods and compositions e.g., other coagulation proteins
  • the invention provides a method of designing the CFXTEN compositions with desired pharmacologic or pharmaceutical properties.
  • the CFXTEN fusion proteins are designed and prepared with various objectives in mind (compared to the CF components not linked to the fusion protein), including improving the therapeutic efficacy for the treatment of coagulation factor-related diseases, disorders, and conditions, enhancing the pharmacokinetic characteristics of the fusion proteins compared to the CF, lowering the dose or frequency of dosing required to achieve a pharmacologic effect, enhancing the pharmaceutical properties, and to enhance the ability of the CF components to remain within the therapeutic window for an extended period of time.
  • the steps in the design and production of the fusion proteins and the inventive compositions include: (1) the selection of CFs (e.g., native proteins, sequences of Tables 1 and 2, analogs or derivatives with activity) to treat the particular disease, disorder or condition; (2) selecting the XTEN that will confer the desired P and physicochemical characteristics on the resulting CFXTEN (e.g., the administration of the CFXTEN composition to a subject results in the fusion protein being maintained within the therapeutic window for a greater period compared to CF not linked to XTEN); (3) establishing a desired N- to C-terminus configuration of the CFXTEN to achieve the desired efficacy or PK parameters; (4) establishing the design of the expression vector encoding the configured CFXTEN; (5) transforming a suitable host with the expression vector; and (6) expression and recovery of the resultant fusion protein.
  • CFs e.g., native proteins, sequences of Tables 1 and 2, analogs or derivatives with activity
  • the XTEN chosen for incorporation generally has at least about 100, or about 144, or about 288, or about 432, or about 576, or about 864, or about 875, or about 912, or about 923 amino acid residues where a single XTEN is to be incorporated into the CFXTEN.
  • the CFXTEN comprises a first XTEN of the foregoing lengths, and at least a second XTEN of about 36, or about 72, or about 144, or about 288, or about 576, or about 864, or about 875, or about 912, or about 923 amino acid residues.
  • a CFXTEN is designed to include XTEN of shorter lengths.
  • the CFXTEN comprises a CF linked to an XTEN having at least about 24, or about 36, or about 48, or about 60, or about 72, or about 84, or about 96 amino acid residues, in which the solubility of the fusion protein under physiologic conditions is at least three-fold greater than the corresponding CF not linked to XTEN, or alternatively, at least four-fold, or five-fold, or six-fold, or seven-fold, or eight-fold, or nine-fold, or at least 10-fold, or at least 20-fold, or at least 30- fold, or at least 50-fold, or at least 60-fold or greater than CF not linked to XTEN.
  • the CF is factor ⁇ . In another embodiment, the CF is factor VII. In another embodiment, the XTEN is a sequence with at least about 80%, or about 90%, or about 95% sequence identity compared to a sequence from Tables 4, and 9-13. WSGR DM. Ref. 32808-726601
  • the invention provides methods of making CFXTEN compositions to improve ease of manufacture, result in increased stability, increased water solubility, and or ease of formulation, as compared to the native CF.
  • the invention includes a method of increasing the water solubility of a CF comprising the step of linking the CF to one or more XTEN such that a higher concentration in soluble form of the resulting CFXTEN can be achieved, under physiologic conditions, compared to the CF in an un-fused state.
  • Factors that contribute to the property of XTEN to confer increased water solubility of CFs when incorporated into a fusion protein include the high solubility of the XTEN fusion partner and the low degree of self-aggregation between molecules of XTEN in solution.
  • the method results in a CFXTEN fusion protein wherein the water solubility is at least about 20%, or at least about 30% greater, or at least about 50% greater, or at least about 75% greater, or at least about 90% greater, or at least about 100% greater, or at least about 150% greater, or at least about 200% greater, or at least about 400% greater, or at least about 600% greater, or at least about 800% greater, or at least about 1000% greater, or at least about 2000% greater, or at least about 4000% greater, or at least about 6000% greater under physiologic conditions, compared to the un-fused CF.
  • the XTEN of the CFXTEN fusion protein is a sequence with at least about 80%, or about 90%, or about 95% sequence identity compared to a sequence from Tables 4, and 9- 13.
  • the invention includes a method of increasing the shelf-life of a CF comprising the step of linking the CF with one or more XTEN selected such that the shelf-life of the resulting CFXTEN is extended compared to the CF in an un-fused state.
  • shelf-life refers to the period of time over which the functional activity of a CF or CFXTEN that is in solution or in some other storage formulation remains stable without undue loss of activity.
  • CF pharmacologic effect or biological activity, such as the ability to bind a receptor or ligand, or substrate, or an enzymatic activity, or to display one or more known functional activities associated with a CF, as known in the art.
  • a CF that degrades or aggregates generally has reduced functional activity or reduced bioavailability compared to one that remains in solution.
  • Factors that contribute to the ability of the method to extend the shelf life of CFs when incorporated into a fusion protein include increased water solubility, reduced self-aggregation in solution, and increased heat stability of the XTEN fusion partner.
  • the low tendency of XTEN to aggregate facilitates methods of formulating pharmaceutical preparations containing higher drug concentrations of CFs, and the heat-stability of XTEN contributes to the property of CFXTEN fusion proteins to remain soluble and functionally active for extended periods.
  • the method results in CFXTEN fusion proteins with "prolonged" or "extended” shelf-life that exhibit greater activity relative to a standard that has been subjected to the same storage and handling conditions.
  • the standard may be the un-fused full- length CF.
  • the method includes the step of formulating the isolated CFXTEN with one or more pharmaceutically acceptable excipients that enhance the ability of the XTEN to retain its unstructured conformation and for the CFXTEN to remain soluble in the formulation for a time that is WSGR Dkt. Ref. 32808-726601 greater than that of the corresponding un-fused CF.
  • the method comprises linking a CF to one or more XTEN selected from Tables 4 and 9- 13 to create a CFXTEN fusion protein results in a solution that retains greater than about 100% of the functional activity, or greater than about 105%, 1 10%, 120%, 130%, 150% or 200% of the functional activity of a standard when compared at a given time point and when subjected to the same storage and handling conditions as the standard, thereby increasing its shelf-life.
  • Shelf-life may also be assessed in terms of functional activity remaining after storage, normalized to functional activity when storage began.
  • CFXTEN fusion proteins of the invention with prolonged or extended shelf-life as exhibited by prolonged or extended functional activity retain about 50% more functional activity, or about 60%, 70%, 80%, or 90% more of the functional activity of the equivalent CF not linked to XTEN when subjected to the same conditions for the same period of time.
  • a CFXTEN fusion protein of the invention comprising coagulation factor fused to one or more XTEN sequences selected from Tables 4 and 9-13 retains about 80% or more of its original activity in solution for periods of up to 2 weeks, or 4 weeks, or 6 weeks or longer under various temperature conditions.
  • the CFXTEN retains at least about 50%, or about 60%, or at least about 70%, or at least about 80%, and most preferably at least about 90% or more of its original activity in solution when heated at 80°C for 10 min. In other embodiments, the CFXTEN retains at least about 50%, preferably at least about 60%, or at least about 70%, or at least about 80%, or alternatively at least about 90% or more of its original activity in solution when heated or maintained at 37°C for about 7 days. In another embodiment, CFXTEN fusion protein retains at least about 80% or more of its functional activity after exposure to a temperature of about 30°C to about 70°C over a period of time of about one hour to about 18 hours.
  • the retained activity of the CFXTEN is at least about two-fold, or at least about three-fold, or at least about four-fold, or at least about five-fold, or at least about six-fold greater at a given time point than that of the corresponding CF not linked to the fusion protein.
  • the present invention provides isolated polynucleic acids encoding CFXTEN chimeric fusion proteins and sequences complementary to polynucleic acid molecules encoding CFXTEN chimeric fusion proteins, including homologous variants thereof.
  • the invention encompasses methods to produce polynucleic acids encoding CFXTEN chimeric fusion proteins and sequences complementary to polynucleic acid molecules encoding CFXTEN chimeric fusion protein, including homologous variants thereof. In general, and as illustrated in FIGS.
  • the methods of producing a polynucleotide sequence coding for a CFXTEN fusion protein and expressing the resulting gene product include assembling nucleotides encoding CF and XTEN, ligating the components in frame, incorporating the encoding gene into an expression vector appropriate for a host cell, transforming the appropriate host cell with the expression vector, and culturing the host cell under conditions causing or permitting the fusion protein to WSGR Dkt. Ref. 32808-726601 be expressed in the transformed host cell, thereby producing the biologically-active CFXTEN
  • polypeptide which is recovered as an isolated fusion protein by standard protein purification methods known in the art. Standard recombinant techniques in molecular biology is used to make the
  • nucleic acid sequences that encode CFXTEN (or its complement) is used to generate recombinant DNA molecules that direct the expression of CFXTEN fusion proteins in appropriate host cells.
  • Several cloning strategies are suitable for performing the present invention, many of which is used to generate a construct that comprises a gene coding for a fusion protein of the CFXTEN composition of the present invention, or its complement.
  • the cloning strategy is used to create a gene that encodes a monomeric CFXTEN that comprises at least a first CF and at least a first XTEN polypeptide, or their complement.
  • the gene comprises a sequence encoding a CF or sequence variant.
  • the cloning strategy is used to create a gene that encodes a monomeric CFXTEN that comprises nucleotides encoding at least a first molecule of CF or its complement and a first and at least a second XTEN or their complement that is used to transform a host cell for expression of the fusion protein of the CFXTEN composition.
  • the genes can further comprise nucleotides encoding spacer sequences that also encode cleavage sequence(s).
  • the XTEN- encoding polynucleotides comprise multiple polynucleotides that encode non-repetitive sequences, or motifs, operably linked in frame and in which the resulting expressed XTEN amino acid sequences are non-repetitive.
  • a construct is first prepared containing the DNA sequence corresponding to CFXTEN fusion protein.
  • DNA encoding the CF of the compositions is obtained from a cDNA library prepared using standard methods from tissue or isolated cells believed to possess CF mRNA and to express it at a detectable level. Libraries is screened with probes containing, for example, about 20 to 100 bases designed to identify the CF gene of interest by hybridization using conventional molecular biology techniques. The best candidates for probes are those that represent sequences that are highly homologous for coagulation factor, and should be of sufficient length and sufficiently unambiguous that false positives are minimized, but may be degenerate at one or more positions.
  • the coding sequence can be obtained using conventional primer extension procedures as described in Sambrook, et al, supra, to WSGR Dkt Ref. 32808-726601 detect precursors and processing intermediates of mRNA that may not have been reverse-transcribed into cDNA.
  • Assays can then be conducted to confirm that hybridizing full-length genes are the desired CF gene(s).
  • DNA can be conveniently obtained from a cDNA library prepared from such sources.
  • the CF encoding gene(s) is also be obtained from a genomic library or created by standard synthetic procedures known in the art (e.g., automated nucleic acid synthesis using, for example one of the methods described in Engels et al. (Agnew. Chem. Int. Ed. Engl., 28:716-734 1989)), using DNA sequences obtained from publicly available databases, patents, or literature references. Such procedures are well known in the art and well described in the scientific and patent literature.
  • sequences can be obtained from Chemical Abstracts Services (CAS) Registry Numbers (published by the American Chemical Society) and/or GenBank Accession Numbers (e.g., Locus ID, NP XXXXX, and XP_XXXXX) Model Protein identifiers available through the National Center for Biotechnology Information (NCBI) webpage, available on the world wide web at
  • ncbi.nlm.nih.gov that correspond to entries in the CAS Registry or GenBank database that contain an amino acid sequence of the protein of interest or of a fragment or variant of the protein.
  • the summary pages associated with each of these CAS and GenBank and GenSeq Accession Numbers as well as the cited journal publications are each incorporated by reference in their entireties, particularly with respect to the amino acid sequences described therein.
  • the CF encoding gene encodes a protein from any one of Table 1 or Table 2, or a fragment or variant thereof.
  • a gene or polynucleotide encoding the CF portion of the subject CFXTEN protein, in the case of an expressed fusion protein that comprises a single CF is then be cloned into a construct, which is a plasmid or other vector under control of appropriate transcription and translation sequences for high level protein expression in a biological system.
  • a second gene or polynucleotide coding for the XTEN is genetically fused to the nucleotides encoding the N- and/or C-terminus of the CF gene by cloning it into the construct adjacent and in frame with the gene(s) coding for the CF.
  • This second step occurs through a ligation or multimerization step.
  • the gene encoding for the XTEN can be made in one or more steps, either fully synthetically or by synthesis combined with enzymatic processes, such as restriction enzyme-mediated cloning, PCR and overlap extension, including methods more fully described in the Examples.
  • the methods disclosed herein can be used, for example, to ligate short sequences of polynucleotides encoding XTEN into longer XTEN genes of a desired length and sequence.
  • the method ligates two or more codon-optimized oligonucleotides encoding XTEN motif or segment sequences of about 9 to 14 amino acids, or about 12 to 20 amino acids, or about 18 to 36 amino acids, or about 48 to about 144 amino acids, WSGR Dkt. Ref. 32808-726601 or about 144 to about 288 or longer, or any combination of the foregoing ranges of motif or segment lengths.
  • the disclosed method is used to multimerize XTEN-encoding sequences into longer sequences of a desired length; e.g., a gene encoding 36 amino acids of XTEN can be dimerized into a gene encoding 72 amino acids, then 144, then 288, etc.
  • XTEN polypeptides can be constructed such that the XTEN-encoding gene has low or virtually no repetitiveness through design of the codons selected for the motifs of the shortest unit being used, which can reduce recombination and increase stability of the encoding gene in the transformed host.
  • Genes encoding XTEN with non-repetitive sequences is assembled from oligonucleotides using standard techniques of gene synthesis.
  • the gene design can be performed using algorithms that optimize codon usage and amino acid composition.
  • a library of relatively short XTEN-encoding polynucleotide constructs is created and then assembled, as illustrated in FIGS. 4 and 5. This can be a pure codon library such that each library member has the same amino acid sequence but many different coding sequences are possible.
  • Such libraries can be assembled from partially randomized
  • oligonucleotides and used to generate large libraries of XTEN segments comprising the sequence motifs.
  • the randomization scheme can be optimized to control amino acid choices for each position as well as codon usage. Exemplary methods to achieve the foregoing are disclosed in the Examples.
  • the invention provides libraries of polynucleotides that encode XTEN sequences that are used to assemble genes that encode XTEN of a desired length and sequence.
  • the XTEN-encoding library constructs comprise polynucleotides that encode polypeptide segments of a fixed length.
  • a library of oligonucleotides that encode motifs of 9-14 amino acid residues can be assembled.
  • libraries of oligonucleotides that encode motifs of 12 amino acids are assembled.
  • the XTEN-encoding sequence segments can be dimerized or multimerized into longer encoding sequences. Dimerization or multimerization can be performed by ligation, overlap extension, PCR assembly or similar cloning techniques known in the art. This process of can be repeated multiple times until the resulting XTEN-encoding sequences have reached the organization of sequence and desired length, providing the XTEN-encoding genes.
  • a library of polynucleotides that encodes e.g., 12 amino acid motifs can be dimerized and/or ligated into a library of polynucleotides that encode 36 amino acids.
  • Libraries encoding motifs of different lengths; e.g., 9-14 amino acid motifs leading to libraries encoding 27 to 42 amino acids are contemplated by the invention.
  • the library of polynucleotides that encode 27 to 42 amino acids, and preferably 36 amino acids (as described in the Examples) can be serially dimerized into a library containing successively longer lengths of
  • libraries are assembled of polynucleotides that encode amino acids that are limited to specific sequence XTEN families; e.g., AD, WSGR Dkt. Ref. 32808-726601
  • libraries comprise sequences that encode two or more of the motif family sequences from Table 3.
  • the names and sequences of representative, non-limiting polynucleotide sequences of libraries that encode 36mers are presented in Tables 9-12, and the methods used to create them are described more fully in the respective Examples.
  • libraries that encode XTEN are constructed from segments of polynucleotide codons linked in a randomized sequence that encode amino acids wherein at least about 80%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% of the codons are selected from the group consisting of condons for glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) amino acids.
  • G glycine
  • A alanine
  • S serine
  • T threonine
  • E glutamate
  • P proline
  • the libraries can be used, in turn, for serial dimerization or ligation to achieve polynucleotide sequence libraries that encode XTEN sequences, for example, of 48, 72, 144, 288, 576, 864, 875, 912, 923, 1318 amino acids, or up to a total length of about 3000 amino acids, as well as intermediate lengths, in which the encoded XTEN can have one or more of the properties disclosed herein, when expressed as a component of a CFXTEN fusion protein.
  • the polynucleotide library sequences may also include additional bases used as "sequencing islands," described more fully below.
  • FIG. 5 is a schematic flowchart of representative, non-limiting steps in the assembly of a XTEN polynucleotide construct and a CFXTEN polynucleotide construct in the embodiments of the invention.
  • Individual oligonucleotides 501 are annealed into sequence motifs 502 such as a 12 amino acid motif ("12-mer"), which is subsequently ligated with an oligo containing Bbsl, and Kpnl restriction sites 503. Additional sequence motifs from a library are annealed to the 12-mer until the desired length of the XTEN gene 504 is achieved.
  • the XTEN gene is cloned into a stuffer vector.
  • the vector optionally encodes a Flag sequence 506 followed by a stuffer sequence that is flanked by Bsal, Bbsl, and Kpnl sites 507 and, in this case, a single CF gene (encoding FIX in this example) 508, resulting in the gene encoding a CFXTEN comprising a single CF 500.
  • a flag sequence 506 followed by a stuffer sequence that is flanked by Bsal, Bbsl, and Kpnl sites 507 and, in this case, a single CF gene (encoding FIX in this example) 508, resulting in the gene encoding a CFXTEN comprising a single CF 500.
  • Table 8 A non-exhaustive list of the XTEN names for polynucleotides encoding XTEN and precursor sequences is provided in Table 8.
  • suitable reporter genes are green fluorescent protein, luciferace, alkaline phosphatase, and beta-galactosidase.
  • One aspect of the invention is to provide polynucleotide sequences encoding the components of the fusion protein wherein the creation of the sequence has undergone codon optimization.
  • codon optimization with the goal of improving expression of the polypeptide compositions and to improve the genetic stability of the encoding gene in the production hosts.
  • codon optimization is of particular importance for XTEN sequences that are rich in glycine or that have very repetitive amino acid sequences. Codon optimization is performed using computer programs (Gustafsson, C, et al. (2004) Trends Biotechnol, 22: 346-53), some of which minimize ribosomal pausing (Coda Genomics Inc.).
  • XTEN sequences one can consider a number of properties. One can minimize the repetitiveness in the encoding DNA sequences. In addition, one can avoid or minimize the use of codons that are rarely used by the production host (e.g. the AGG and AGA arginine codons and one leucine codon in E. coli). In the case of E.
  • coli two glycine codons, GGA and GGG, are rarely used in highly expressed proteins.
  • codon optimization of the gene encoding XTEN sequences can be very desirable.
  • DNA sequences that have a high level of glycine tend to have a high GC content that can lead to instability or low expression levels.
  • codons such that the GC-content of XTEN-encoding sequence is suitable for the production organism that will be used to manufacture the XTEN.
  • the full-length XTEN-encoding gene comprises one or more sequencing islands.
  • sequencing islands are short-stretch sequences that are distinct from the XTEN library construct sequences and that include a restriction site not present or expected to be present in the full- length XTEN-encoding gene.
  • a sequencing island is the sequence
  • a sequencing island is the sequence
  • polynucleotide libraries are constructed using the disclosed methods wherein all members of the library encode the same amino acid sequence but where codon usage for the respective amino acids in the sequence is varied. Such libraries can be screened for highly expressing and genetically stable members that are particularly suitable for the large-scale production of XTEN- containing products.
  • the initial library of short XTEN sequences allows some variation in amino acid sequence. For instance one can randomize some codons such that a number of hydrophilic amino acids can occur in a particular position.
  • the gene that encodes the XTEN of desired length and properties is selected, it is genetically fused at the desired location to the nucleotides encoding the CF gene(s) by cloning it into the construct adjacent and in frame with the gene coding for CF, or alternatively between nucleotides encoding adjacent domains of the CF, or alternatively within a sequence encoding a given CF domain, or alternatively in frame with nucleotides encoding a spacer/cleavage sequence linked to a terminal XTEN.
  • the invention provides various permutations of the foregoing, depending on the CFXTEN to be encoded.
  • a gene encoding a CFXTEN fusion protein comprising a CF and two XTEN such as WSGR Dkt. Ref. 32808-726601 embodied by formula VI, as depicted above, the gene would have polynucleotides encoding CF, encoding two XTEN, which can be identical or different in composition and sequence length.
  • the CF polynucleotides would encode coagulation factor and the polynucleotides encoding the C-terminus XTEN would encode AE864 and the polynucleotides encoding an internal XTEN adjacent to the C-terminus of EGF2 would encode AE144.
  • the step of cloning the CF genes into the XTEN construct can occur through a ligation or multimerization step, as shown in FIG. 32.
  • the constructs encoding CFXTEN fusion proteins can be designed in different configurations of the components XTEN, CF, and spacer sequences, such as the configurations of formulae I- VI.
  • the construct comprises polynucleotide sequences complementary to, or those that encode a monomeric polypeptide of components in the following order (5' to 3') CF and XTEN.
  • the construct comprises polynucleotide sequences complementary to, or those that encode a monomeric polypeptide of components in the following order (5' to 3') CF, spacer sequence, and XTEN.
  • the spacer polynucleotides can optionally comprise sequences encoding cleavage sequences. As will be apparent to those of skill in the art, other permutations or multimers of the foregoing are possible.
  • the invention also encompasses polynucleotides comprising XTEN-encoding polynucleotide variants that have a high percentage of sequence identity compared to (a) a polynucleotide sequence from Table 8, or (b) sequences that are complementary to the polynucleotides of (a).
  • a polynucleotide with a high percentage of sequence identity is one that has at least about an 80% nucleic acid sequence identity, alternatively at least about 81%, alternatively at least about 82%, alternatively at least about 83%, alternatively at least about 84%, alternatively at least about 85%, alternatively at least about 86%, alternatively at least about 87%, alternatively at least about 88%, alternatively at least about 89%, alternatively at least about 90%, alternatively at least about 91% , alternatively at least about 92%, alternatively at least about 93%, alternatively at least about 94%, alternatively at least about 95%, alternatively at least about 96%, alternatively at least about 97%, alternatively at least about 98%, and alternatively at least about 99% nucleic acid sequence identity compared to (a) or (b) of the foregoing, or that can hybridize with the target polynucleotide or its complement under stringent conditions.
  • Homology, sequence similarity or sequence identity of nucleotide or amino acid sequences may also be determined conventionally by using known software or computer programs such as the BestFit or Gap pairwise comparison programs (GCG Wisconsin Package, Genetics Computer Group, 575 Science Drive, Madison, Wis. 53711). BestFit uses the local homology algorithm of Smith and
  • Nucleic acid sequences that are "complementary" are those that are capable of base-pairing according to the standard Watson-Crick complementarity rules. As used herein, the term
  • complementary sequences means nucleic acid sequences that are substantially complementary, as may be assessed by the same nucleotide comparison set forth above, or as defined as being capable of hybridizing to the polynucleotides that encode the CFXTEN sequences under stringent conditions, such as those described herein.
  • the resulting polynucleotides encoding the CFXTEN chimeric fusion proteins can then be individually cloned into an expression vector.
  • the nucleic acid sequence is inserted into the vector by a variety of procedures.
  • DNA is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art.
  • Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan. Such techniques are well known in the art and well described in the scientific and patent literature.
  • the vector may, for example, be in the form of a plasmid, cosmid, viral particle, or phage that may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced.
  • the vector may be an autonomously replicating vector, i.e., a vector, which exists as an
  • extrachromosomal entity the replication of which is independent of chromosomal replication
  • the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
  • the invention provides for the use of plasmid vectors containing replication and control sequences that are compatible with and recognized by the host cell, and are operably linked to the CFXTEN gene for controlled expression of the CFXTEN fusion proteins.
  • the vector ordinarily carries a replication site, as well as sequences that encode proteins that are capable of providing phenotypic selection in transformed cells.
  • Such vector sequences are well known for a variety of bacteria, yeast, and viruses.
  • Useful expression vectors that can be used include, for example, segments of chromosomal, non- chromosomal and synthetic DNA sequences.
  • “Expression vector” refers to a DNA construct containing a DNA sequence that is operably linked to a suitable control sequence capable of effecting the expression of the DNA encoding the fusion protein in a suitable host. The requirements are that the vectors are replicable and viable in the host cell of choice. Low- or high-copy number vectors may be used as desired.
  • Suitable vectors include, but are not limited to, derivatives of SV40 and pcDNA and known bacterial plasmids such as col EI, pCRl, pBR322, pMal-C2, pET, pGEX as described by Smith, et al., Gene 57:31-40 (1988), pMB9 and derivatives thereof, plasmids such as RP4, phage DNAs such as the numerous derivatives of phage I such as NM98 9, as well as other phage DNA such as M 13 and filamentous single stranded phage DNA; yeast plasmids such as the 2 micron plasmid or derivatives of WSGR Dkt Ref.
  • plasmids such as RP4, phage DNAs such as the numerous derivatives of phage I such as NM98 9, as well as other phage DNA such as M 13 and filamentous single stranded phage DNA; yeast plasmids such as the 2 micron plasmid or
  • yeast expression systems that can also be used in the present invention include, but are not limited to, the non-fusion pYES2 vector (Invitrogen), the fusion pYESHisA, B, C (Invitrogen), pRS vectors and the like.
  • control sequences of the vector include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences that control termination of transcription and translation.
  • the promoter may be any DNA sequence, which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.
  • Suitable promoters for directing the transcription of the DNA encoding the CF polypeptide variant in mammalian cells are the SV40 promoter (Subramani et al., Mol. Cell. Biol. 1 (1981), 854-864), the MT-1 (metallothionein gene) promoter (Palmiter et al., Science 222 (1983), 809- 814), the CMV promoter (Boshart et al., Cell 41 :521-530, 1985) or the adenovirus 2 major late promoter (Kaufman and Sharp, Mol. Cell. Biol, 2: 1304-1319, 1982).
  • the vector may also carry sequences such as UCOE (ubiquitous chromatin opening elements).
  • suitable promoters for use in filamentous fungus host cells are, for instance, the ADH3 promoter or the tpiA promoter.
  • suitable promoters are those derived from the gene encoding A. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. niger neutral a- amylase, A. niger acid stable ⁇ -amylase, A. niger or A. awamoriglucoamylase (gluA), Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase or A. nidulans acetamidase.
  • Preferred are the TAKA-amylase and gluA promoters.
  • Promoters suitable for use in expression vectors with prokaryotic hosts include the ⁇ - lactamase and lactose promoter systems [Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature, 281 :544 (1979)], alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36,776], and hybrid promoters such as the tac promoter [deBoer et al., Proc. Natl. Acad. Sci.
  • Promoters for use in bacterial systems can also contain a Shine-Dalgarno (S.D.) sequence, operably linked to the DNA encoding CFXTEN polypeptides.
  • S.D. Shine-Dalgarno
  • the invention contemplates use of other expression systems including, for example, a baculovirus expression system with both non-fusion transfer vectors, such as, but not limited to pVL941 Summers, et al., Virology 84:390-402 (1978)), pVL1393 (Invitrogen), pVL1392 (Summers, et al., Virology 84:390- 402 (1978) and Invitrogen) and pBlueBacIII (Invitrogen), and fusion transfer vectors such as, but not limited to, pAc7 00 (Summers, et al., Virology 84:390-402 (1978)), pAc701 and pAc70-2 (same as pAc700, with different reading frames), pAc360 Invitrogen) and pBlueBacHisA, B, C (;
  • Examples of suitable promoters for directing the transcription of the DNA encoding the CF polypeptide variant in mammalian cells are the CMV promoter (Boshart et al., Cell 41 :521-530, 1985), the SV40 promoter (Subramani et al., Mol Cell. Biol. 1 (1981), 854-864), the MT-1 (metal lothionein gene) promoter (Palmiter et al., Science 222 (1983), 809-814), the adenovirus 2 major late promoter (Kaufman and Sharp, Mol. Cell. Biol, 2: 1304-1319, 1982).
  • the vector may also carry sequences such as UCOE (ubiquitous chromatin opening elements).
  • suitable promoters for use in filamentous fungus host cells are, for instance, the ADH3 promoter or the tpiA promoter.
  • the DNA sequences encoding the CFXTEN may also, if necessary, be operably connected to a suitable terminator, such as the hGH terminator (Palmiter et al., Science 222, 1983, pp. 809-814) or the TPI1 terminators (Alber and Kawasaki, J. Mol. Appl. Gen. 1 , 1982, pp. 419-434) or ADH3 (McKnight et al., The EMBO J. 4, 1985, pp. 2093-2099).
  • Expression vectors may also contain a set of RNA splice sites located downstream from the promoter and upstream from the insertion site for the CFXTEN sequence itself, including splice sites obtained from adenovirus.
  • polyadenylation signal located downstream of the insertion site.
  • Particularly preferred polyadenylation signals include the early or late polyadenylation signal from SV40 (Kaufman and Sharp, ibid.), the polyadenylation signal from the adenovirus 5 Elb region, the hGH terminator (DeNoto et al. Nucl. Acids Res. 9:3719-3730, 1981).
  • the expression vectors may also include a noncoding viral leader sequence, such as the adenovirus 2 tripartite leader, located between the promoter and the RNA splice sites; and enhancer sequences, such as the SV40 enhancer.
  • a secretory signal sequence (a.k.a., a leader sequence, a prepro sequence, or a pre sequence) may be included in the recombinant vector.
  • the secretory signal sequence is operably linked to the DNA sequences encoding the CFXTEN, usually positioned 5' to the DNA sequence encoding the CFXTEN fusion protein.
  • the secretory signal sequence may be that, normally associated with the protein or may be from a gene encoding another secreted protein. Non-limiting examples include OmpA, PhoA, and DsbA for E.
  • coli expression ppL-alpha, DEX4, invertase signal peptide, acid phosphatase signal peptide, CPY, or INU1 for yeast expression, and IL2L, SV40, IgG kappa and IgG lambda for mammalian expression.
  • Signal sequences are typically proteolytically removed from the protein during the translocation and secretion process, generating a defined N-terminus. Methods are disclosed in Arnau, et al., Protein Expression and Purification 48: 1-13 (2006).
  • the invention provides constructs and methods of making constructs comprising an polynucleotide sequence optimized for expression that encodes at least about 20 to about 60 amino acids with XTEN characteristics that can be included at the N-terminus of an XTEN carrier encoding sequence (in other words, the polynucleotides encoding the 20-60 encoded optimized amino acids are linked in frame to polynucleotides encoding an XTEN component that is N-terminal to CF) to promote the initiation of translation to allow for expression of XTEN fusions at the N-terminus of proteins without the presence of a helper domain.
  • the sequence does not require subsequent cleavage, thereby reducing the number of steps to manufacture XTEN-containing compositions.
  • the optimized N-terminal sequence has attributes of an unstructured protein, but may include nucleotide bases encoding amino acids selected for their ability to promote initiation of translation and enhanced expression.
  • the optimized polynucleotide encodes an XTEN sequence with at least about 90% sequence identity compared to AE912. In another embodiment of the foregoing, the optimized polynucleotide encodes an XTEN sequence with at least about 90% sequence identity compared to AM923.
  • the optimized polynucleotide encodes an XTEN sequence with at least about 90% sequence identity compared to AE48. In another embodiment of the foregoing, the optimized polynucleotide encodes an XTEN sequence with at least about 90% sequence identity compared to AM48. In one embodiment, the optimized polynucleotide NTS comprises a sequence that exhibits at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, sequence identity compared to a sequence or its complement selected from AE 48: 5'-
  • a chimeric DNA molecule coding for a monomeric CFXTEN fusion protein is generated within the construct.
  • this chimeric DNA molecule may be transferred or cloned into another construct that is a more appropriate expression vector.
  • a host cell capable of expressing the chimeric DNA molecule can be transformed with the chimeric DNA molecule.
  • Examples of mammalian cell lines for use in the present invention are the COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), BHK-21 (ATCC CCL 10)) and BHK-293 (ATCC CRL 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977), BHK-570 cells (ATCC CRL 10314), CHO-K1 (ATCC CCL 61), WSGR Dkt Ref. 32808-726601
  • CHO-S Invitrogen 1 1619-012), and 293-F (Invitrogen R790-7).
  • a tk ⁇ tsl 3 BHK cell line is also available from the ATCC under accession number CRL 1632.
  • a number of other cell lines may be used within the present invention, including Rat Hep I (Rat hepatoma; ATCC CRL 1600), Rat Hep II (Rat hepatoma; ATCC CRL 1548), TCMK (ATCC CCL 139), Human lung (ATCC HB 8065), NCTC 1469 (ATCC CCL 9.1), CHO (ATCC CCL 61) and DUKX cells (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220, 1980).
  • yeasts cells include cells of Saccharomyces spp. or Schizosaccharomyces spp., in particular strains of Saccharomyces cerevisiae or Saccharomyces kluyveri .
  • Methods for transforming yeast cells with heterologous DNA and producing heterologous polypeptides there from are described, e.g. in U.S. Pat. No. 4,599,3 1 1, U.S. Pat. No. 4,931,373, U.S. Pat. No. 4,870,008, 5,037,743, and U.S. Pat. No. 4,845,075, all of which are hereby incorporated by reference.
  • Transformed cells are selected by a phenotype determined by a selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient, e.g. leucine.
  • a preferred vector for use in yeast is the POT1 vector disclosed in U.S. Pat. No. 4,931 ,373.
  • the DNA sequences encoding the CFXTEN may be preceded by a signal sequence and optionally a leader sequence, e.g. as described above.
  • suitable yeast cells are strains of Kluyveromyces, such as K. lactis, Hansenula , e.g. H. polymorpha , or Pichia , e.g. P. pastoris (cf. Gleeson et al., J. Gen.
  • Examples of other fungal cells are cells of filamentous fungi, e.g. Aspergillus spp., Newospora spp., Fusarium spp. or Trichoderma spp., in particular strains of A. oryzae, A. nidulans ox A. niger .
  • Aspergillus spp. for the expression of proteins is described in, e.g., EP 272 277, EP 238 023, EP 184 438
  • the transformation of F. oxysporum may, for instance, be carried out as described by Malardier et al., 1989 , Gene 78: 147-156.
  • the transformation of Trichoderma spp. may be performed for instance as described in EP 244 234.
  • suitable cells that can be used in the present invention include, but are not limited to, prokaryotic host cells strains such as Escherichia coli, (e.g., strain DH5-a), Bacillus subtilis, Salmonella typhimurium, or strains of the genera of Pseudomonas, Streptomyces and Staphylococcus.
  • prokaryotic host cells strains such as Escherichia coli, (e.g., strain DH5-a), Bacillus subtilis, Salmonella typhimurium, or strains of the genera of Pseudomonas, Streptomyces and Staphylococcus.
  • suitable prokaryotes include those from the genera: Actinoplanes; Archaeoglobus;
  • Thermoplasma Thermoplasma; and Vibrio.
  • a gene that confers a selectable phenotype is generally introduced into cells along with the gene or cDNA of interest.
  • selectable markers include genes that confer resistance to drugs such as neomycin, hygromycin, puromycin, zeocin, and methotrexate.
  • the selectable marker may be an amplifiable selectable marker.
  • a preferred amplifiable selectable marker is a dihydrofolate reductase (DHFR) sequence.
  • DHFR dihydrofolate reductase
  • Further examples of selectable markers are well known to one of skill in the art and include reporters such as enhanced green fluorescent protein (EGFP), beta-galactosidase ( ⁇ -gal) or
  • CAT chloramphenicol acetyltransferase
  • Selectable markers may be introduced into the cell on a separate plasmid at the same time as the gene of interest, or they may be introduced on the same plasmid. If, on the same plasmid, the selectable marker and the gene of interest may be under the control of different promoters or the same promoter, the latter arrangement producing a dicistronic message. Constructs of this type are known in the art (for example, Levinson and Simonsen, U.S. Pat. No. 4,713,339). It may also be advantageous to add additional DNA, known as "carrier DNA,” to the mixture that is introduced into the cells.
  • carrier DNA additional DNA
  • the cells After the cells have taken up the DNA, they are grown in an appropriate growth medium, typically 1-2 days, to begin expressing the gene of interest.
  • appropriate growth medium means a medium containing nutrients and other components required for the growth of cells and the expression of the CFXTEN of interest.
  • Media generally include a carbon source, a nitrogen source, essential amino acids, essential sugars, vitamins, salts, phospholipids, protein and growth factors.
  • the medium will contain vitamin K, preferably at a concentration of about 0.1 ⁇ ⁇ to about 5 ⁇ g/ml.
  • Drug selection is then applied to select for the growth of cells that are expressing the selectable marker in a stable fashion.
  • the drug concentration may be increased to select for an increased copy number of the cloned sequences, thereby increasing expression levels.
  • Clones of stably transfected cells are then screened for expression of the CF polypeptide variant of interest.
  • the transformed or transfected host cell is then cultured in a suitable nutrient medium under conditions permitting expression of the CF polypeptide variant after which the resulting peptide may be recovered from the culture.
  • the medium used to culture the cells may be any conventional medium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared according to WSGR Dkt. Ref. 32808-726601 published recipes (e.g. in catalogues of the American Type Culture Collection).
  • the culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
  • Gene expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA [Thomas, Proc. Natl. Acad. Sci. USA, 77:5201 -5205 (1980)], dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein.
  • antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn may be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.
  • Gene expression may be measured by immunological of fluorescent methods, such as immunohistochemical staining of cells or tissue sections and assay of cell culture or body fluids or the detection of selectable markers, to quantitate directly the expression of gene product.
  • Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal. Conveniently, the antibodies may be prepared against a native sequence CF polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to CF and encoding a specific antibody epitope.
  • selectable markers are well known to one of skill in the art and include reporters such as enhanced green fluorescent protein (EGFP), beta-galactosidase ( ⁇ -gal) or chloramphenicol acetyltransferase (CAT).
  • Expressed CFXTEN polypeptide product(s) may be purified via methods known in the art or by methods disclosed herein. Procedures such as gel filtration, affinity purification (e.g., using an anti-CF antibody column), salt fractionation, ion exchange chromatography, size exclusion chromatography, hydroxyapatite adsorption chromatography, hydrophobic interaction chromatography and gel electrophoresis may be used; each tailored to recover and purify the fusion protein produced by the respective host cells. Additional purification may be achieved by conventional chemical purification means, such as high performance liquid chromatography. Some expressed CFXTEN may require refolding during isolation and purification. Methods of purification are described in Robert . Scopes, Protein Purification: Principles and Practice, Charles R. Castor (ed.), Springer- Verlag 1994, and
  • the CFXTEN fusion proteins of the invention are substantially pure.
  • the CFXTEN of the invention is purified to at least about 90 to 95% homogeneity, preferably to at least about 98% homogeneity. Purity may be assessed by, e.g., gel electrophoresis, HPLC, and amino-terminal amino acid sequencing..
  • the present invention provides pharmaceutical compositions comprising CFXTEN.
  • the pharmaceutical composition comprises the CFXTEN fusion protein and at least one pharmaceutically acceptable carrier.
  • CFXTEN polypeptides of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the polypeptide is combined in admixture with a pharmaceutically acceptable carrier vehicle, such as aqueous solutions or buffers, pharmaceutically acceptable suspensions and emulsions.
  • a pharmaceutically acceptable carrier vehicle such as aqueous solutions or buffers, pharmaceutically acceptable suspensions and emulsions.
  • non-aqueous solvents include propyl ethylene glycol, polyethylene glycol and vegetable oils.
  • Therapeutic formulations are prepared for storage by mixing the active ingredient having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers, as described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980), in the form of lyophilized formulations or aqueous solutions.
  • the pharmaceutical compositions can be administered orally, intranasally, parenterally or by inhalation therapy, and may take the form of tablets, lozenges, granules, capsules, pills, ampoules, suppositories or aerosol form. They may also take the form of suspensions, solutions and emulsions of the active ingredient in aqueous or nonaqueous diluents, syrups, granulates or powders. In addition, the pharmaceutical compositions can also contain other pharmaceutically active compounds or a plurality of compounds of the invention.
  • compositions may be administered for therapy by any suitable route including oral, rectal, nasal, topical (including transdermal, aerosol, buccal and sublingual), vaginal, parenteral (including subcutaneous, subcutaneous by infusion pump, intramuscular, intravenous and intradermal), intravitreal, and pulmonary. It will also be appreciated that the preferred route will vary with the condition and age of the recipient, and the disease being treated.
  • the pharmaceutical composition is administered subcutaneously.
  • the composition may be supplied as a lyophilized powder to be reconstituted prior to administration.
  • the composition may also be supplied in a liquid form, which can be administered directly to a patient.
  • the composition is supplied as a liquid in a pre-filled syringe such that a patient can easily self-administer the composition.
  • Extended release formulations useful in the present invention may be oral formulations comprising a matrix and a coating composition.
  • suitable matrix materials may include waxes (e.g., camauba, bees wax, paraffin wax, ceresine, shellac wax, fatty acids, and fatty alcohols), oils, hardened oils or fats (e.g., hardened rapeseed oil, castor oil, beef tallow, palm oil, and soya bean oil), and polymers (e.g., hydroxypropyl cellulose, polyvinylpyrrolidone, hydroxypropyl methyl cellulose, and polyethylene glycol).
  • waxes e.g., camauba, bees wax, paraffin wax, ceresine, shellac wax, fatty acids, and fatty alcohols
  • oils hardened oils or fats (e.g., hardened rapeseed oil, castor oil, beef tallow, palm oil, and soya bean oil)
  • polymers e.g.,
  • Suitable matrix tabletting materials are microcrystalline cellulose, powdered cellulose, hydroxypropyl cellulose, ethyl cellulose, with other carriers, and fillers. Tablets may also contain granulates, coated powders, or pellets. Tablets may also be multi-layered. Multi-layered tablets are especially preferred when the active ingredients have markedly different pharmacokinetic profiles.
  • the finished tablet may be coated or uncoated.
  • the coating composition may comprise an insoluble matrix polymer and/or a water soluble material.
  • Water soluble materials can be polymers such as polyethylene glycol, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinylpyrrolidone, polyvinyl alcohol, or monomeric materials such as sugars (e.g., lactose, sucrose, fructose, mannitol and the like), salts (e.g., sodium chloride, potassium chloride and the like), organic acids (e.g., fumaric acid, succinic acid, lactic acid, and tartaric acid), and mixtures thereof.
  • an enteric polymer may be incorporated into the coating composition.
  • Suitable enteric polymers include hydroxypropyl methyl cellulose, acetate succinate, hydroxypropyl methyl cellulose, phthalate, polyvinyl acetate phthalate, cellulose acetate phthalate, cellulose acetate trimellitate, shellac, zein, and polymethacrylates containing carboxyl groups.
  • the coating composition may be plasticised by adding suitable plasticisers such as, for example, diethyl phthalate, citrate esters, polyethylene glycol, glycerol, acetylated glycerides, acetylated citrate esters, dibutylsebacate, and castor oil.
  • the coating composition may also include a filler, which can be an insoluble material such as silicon dioxide, titanium dioxide, talc, kaolin, alumina, starch, powdered cellulose, MCC, or polacrilin potassium.
  • a filler which can be an insoluble material such as silicon dioxide, titanium dioxide, talc, kaolin, alumina, starch, powdered cellulose, MCC, or polacrilin potassium.
  • the coating composition may be applied as a solution or latex in organic solvents or aqueous solvents or mixtures thereof. Solvents such as water, lower alcohol, lower chlorinated hydrocarbons, ketones, or mixtures thereof may be used.
  • compositions of the invention may be formulated using a variety of excipients.
  • Suitable excipients include microcrystalline cellulose (e.g. Avicel PHI 02, Avicel PHI 01), polymethacrylate, poly(ethyl acrylate, methyl methacrylate, trimethylammonioethyl methacrylate chloride) (such as Eudragit RS-30D), hydroxypropyl methylcellulose (Methocel K100M, Premium CR Methocel 100M, Methocel E5, Opadry®), magnesium stearate, talc, triethyl citrate, aqueous ethylcellulose dispersion (Surelease®), and protamine sulfate.
  • microcrystalline cellulose e.g. Avicel PHI 02, Avicel PHI 01
  • polymethacrylate poly(ethyl acrylate, methyl methacrylate, trimethylammonioethyl methacrylate chloride)
  • the slow release agent may also comprise a carrier, which can comprise, for example, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents.
  • Pharmaceutically acceptable salts can also be used in these slow release agents, for example, mineral salts such as hydrochlorides, hydrobromides, phosphates, or sulfates, as well as the salts of organic acids such as acetates, proprionates, malonates, or benzoates.
  • the composition may also contain liquids, such as water, saline, glycerol, and ethanol, as well as substances such as wetting agents, emulsifying agents, or pH buffering agents. Liposomes may also be used as a carrier.
  • compositions of the present invention are encapsulated in liposomes, which have demonstrated utility in delivering beneficial active agents in a controlled manner over prolonged periods of time.
  • Liposomes are closed bilayer membranes containing an entrapped aqueous volume. Liposomes may also be unilamellar vesicles possessing a single membrane bilayer or multilamellar vesicles with multiple membrane bilayers, each separated from the next by an aqueous layer.
  • the structure of the resulting membrane bilayer is such that the hydrophobic (non-polar) tails of the lipid are oriented toward the center of the bilayer while the hydrophilic (polar) heads orient towards the aqueous phase.
  • the liposome may be coated with a flexible water soluble polymer that avoids uptake by the organs of the mononuclear phagocyte system, primarily the liver and spleen.
  • a flexible water soluble polymer that avoids uptake by the organs of the mononuclear phagocyte system, primarily the liver and spleen.
  • Suitable hydrophilic polymers for surrounding the liposomes include, without limitation, PEG, polyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline, polyethyloxazoline,
  • polyhydroxypropyloxazoline polyhydroxypropylmethacrylamide, polymethacrylamide,
  • Liposomes may be comprised of any lipid or lipid combination known in the art.
  • the vesicle-forming lipids may be naturally-occurring or synthetic lipids, including phospholipids, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylserine,
  • the vesicle-forming lipids may also be glycolipids, cerebrosides, or cationic lipids, such as l,2-dioleyloxy-3-(trimethyIamino) propane (DOTAP); N-[l-(2,3,-ditetradecyloxy)propyl]-N,N- dimethyl-N-hydroxyethylammonium bromide (DMRIE); N-[l [(2,3,-dioleyloxy)propyl]-N,N-dimethyl- N-hydroxy ethylammonium bromide (DORIE); N-[ l -(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA); 3 [N-(
  • a desired property is that the formulation be supplied in a form that can pass through a 25, 28, 30, 31 , 32 gauge needle for intravenous, intramuscular, intraarticular, or subcutaneous administration.
  • transdermal formulations can be performed using methods also known in the art, including those described generally in, e.g., U.S. Pat. Nos. 5, 186,938 and 6, 183,770, 4,861 ,800, 6,743,21 1 , 6,945,952, 4,284,444, and WO 89/09051 , incorporated herein by reference in their entireties.
  • a transdermal patch is a particularly useful embodiment with polypeptides having absorption problems. Patches can be made to control the release of skin-permeable active ingredients over a 12 hour, 24 hour, 3 day, and 7 day period. In one example, a 2-fold daily excess of a polypeptide of the present invention is placed in a non-volatile fluid.
  • compositions of the invention are provided in the form of a viscous, non-volatile liquid.
  • the penetration through skin of specific formulations may be measures by standard methods in the art (for example, Franz et al., J. Invest. Derm. 64: 194- 1 5 ( 1975)).
  • suitable WSGR Dkt Ref. 32808-726601 patches are passive transfer skin patches, iontophoretic skin patches, or patches with microneedles such as Nicoderm.
  • the composition may be delivered via intranasal, buccal, or sublingual routes to the brain to enable transfer of the active agents through the olfactory passages into the CNS and reducing the systemic administration.
  • Devices commonly used for this route of administration are included in U.S. Pat. No. 6,715,485.
  • Compositions delivered via this route may enable increased CNS dosing or reduced total body burden reducing systemic toxicity risks associated with certain drugs.
  • Preparation of a pharmaceutical composition for delivery in a subdermally implantable device can be performed using methods known in the art, such as those described in, e.g., U.S. Pat. Nos. 3,992,518; 5,660,848; and 5,756,1 15.
  • Osmotic pumps may be used as slow release agents in the form of tablets, pills, capsules or implantable devices.
  • Osmotic pumps are well known in the art and readily available to one of ordinary skill in the art from companies experienced in providing osmotic pumps for extended release drug delivery. Examples are ALZA's DUROSTM; ALZA's OROSTM; Osmotica Pharmaceutical's OsmodexTM system; Shire Laboratories' EnSoTrolTM system; and AlzetTM.
  • Patents that describe osmotic pump technology are U.S. Pat. Nos. 6,890,918; 6,838,093; 6,814,979; 6,713,086; 6,534,090; 6,514,532;
  • Syringe pumps may also be used as slow release agents.
  • Such devices are described in U.S. Pat. Nos. 4,976,696; 4,933,185; 5,017,378; 6,309,370; 6,254,573; 4,435,173; 4,398,908; 6,572,585; 5,298,022; 5,176,502; 5,492,534; 5,318,540; and 4,988,337, the contents of which are incorporated herein by reference.
  • One skilled in the art considering both the disclosure of this invention and the disclosures of these other patents could produce a syringe pump for the extended release of the compositions of the present invention.
  • the invention provides a kit to facilitate the use of the CFXTEN
  • the kit comprises the pharmaceutical composition provided herein, a label identifying the pharmaceutical composition, and an instruction for storage, reconstitution and/or administration of the pharmaceutical compositions to a subject.
  • the kit comprises, preferably: (a) an amount of a CFXTEN fusion protein composition sufficient to treat a disease, condition or disorder upon administration to a subject in need thereof; and (b) an amount of a pharmaceutically acceptable carrier; together in a formulation ready for injection or for reconstitution with sterile water, buffer, or dextrose; together with a label identifying the CFXTEN drug and storage and handling conditions, and a sheet of the approved indications for the drug, instructions for the reconstitution and/or administration of the WSGR Dkt. Ref. 32808-726601
  • the kit can comprise a second container that can carry a suitable diluent for the CFXTEN composition, the use of which will provide the user with the appropriate concentration of CFXTEN to be delivered to the subject.
  • Example 1 Construction of XTEN_AD36 motif segments
  • pCW0359 was constructed based on a pET vector and that includes a T7 promoter. pCW0359 encodes a cellulose binding domain
  • CBD CBD
  • TEV protease recognition site followed by a stuffer sequence that is flanked by Bsal, Bbsl, and Kpnl sites.
  • the Bsal and Bbsl sites were inserted such that they generate compatible overhangs after digestion.
  • the stuffer sequence is followed by a truncated version of the GFP gene and a His tag.
  • the stuffer sequence contains stop codons and thus E. coli cells carrying the stuffer plasmid pCW0359 form non-fluorescent colonies.
  • the stuffer vector pCW0359 was digested with Bsal and Kpnl to remove the stuffer segment and the resulting vector fragment was isolated by agarose gel purification.
  • the sequences were designated XTEN AD36, reflecting the AD family of motifs. Its segments have the amino acid sequence [X] 3 where X is a 12mer peptide with the sequences: GESPGGSSGSES, GSEGSSGPGESS,
  • GSSESGSSEGGP GSGGEPSESGSS.
  • the insert was obtained by annealing the following pairs of phosphorylated synthetic oligonucleotide pairs:
  • AD2rev ACCTGARGAYTCRCCHGGACCRGAAGAACCTTCGCT
  • AD3for AGGTTCYTCYGAAAGCGGTTCTTCYGARGGYGGTCC
  • AD4for AGGTTCYGGTGGYGAACCDTCYGARTCTGGTAGCTC
  • pr_3KpnIstopperRev CCTCGAGTGAAGACGA.
  • the annealed oligonucleotide pairs were ligated, which resulted in a mixture of products with varying length that represents the varying number of 12mer repeats ligated to one Bbsl/Kpnl segment.
  • the products corresponding to the length of 36 amino acids were isolated from the mixture by preparative agarose gel electrophoresis and ligated into the Bsal/Kpnl digested stuffer vector pCW0359.
  • the XTEN sequence was designated XTEN_AE36. Its segments have the amino acid sequence [X] 3 where X is a 12mer peptide with the sequence: GSPAGSPTSTEE, GSEPATSGSE TP, GTSESA TPESGP, or GTSTEPSEGSAP.
  • the insert was obtained by annealing the following pairs of phosphorylated synthetic oligonucleotide pairs:
  • pr_3KpnIstopperRev CCTCGAGTGAAGACGA.
  • the annealed oligonucleotide pairs were ligated, which resulted in a mixture of products with varying length that represents the varying number of 12mer repeats ligated to one Bbsl/Kpnl segment.
  • the products corresponding to the length of 36 amino acids were isolated from the mixture by preparative agarose gel electrophoresis and ligated into the Bsal/Kpnl digested stuffer vector pCW0359.
  • Most of the clones in the resulting library designated LCW0402 showed green fluorescence after induction which shows that the sequence of XTEN AE36 had been ligated in frame with the GFP gene and most sequences of XTEN AE36 show good expression.
  • a codon library encoding sequences of 36 amino acid length was constructed. The sequences were designated XTEN AF36. Its segments have the amino acid sequence [X]3 where X is a 12mer peptide with the sequence: GSTSESPSGTAP, GTSTPESGSASP, GTSPSGESSTAP, or
  • the insert was obtained by annealing the following pairs of phosphorylated synthetic oligonucleotide pairs:
  • AFlrev ACCTGGWGCRGTRCCAGAMGGRGATTCGCTRGTAGA
  • AF2rev ACCTGGAGAWGCRGARCCGCTTTCMGGRGTAGARGT
  • AF3rev ACCTGGWGCRGTAGAAGATTCRCCGCTMGGRGARGT
  • AF4rev ACCTGGRCCMGGAGATTCWGCRGTAGAGCTRGTRGA
  • pr_3 pnIstopperRev CCTCGAGTGAAGACGA.
  • the annealed oligonucleotide pairs were ligated, which resulted in a mixture of products with varying length that represents the varying number of 12mer repeats ligated to one Bbsl/Kpnl segment
  • the products corresponding to the length of 36 amino acids WSGR Dkt. Ref. 32808-726601 were isolated from the mixture by preparative agarose gel electrophoresis and ligated into the Bsal/Kpnl digested stuffer vector pCW0359.
  • Most of the clones in the resulting library designated LCW0403 showed green fluorescence after induction which shows that the sequence of XTEN AF36 had been ligated in frame with the GFP gene and most sequences of XTEN AF36 show good expression.
  • a codon library encoding sequences of 36 amino acid length was constructed. The sequences were designated XTEN_AG36. Its segments have the amino acid sequence [X] 3 where X is a 12mer peptide with the sequence: GTPGSGTASSSP, GSSTPSGATGSP, GSSPSASTGTGP, or GASPGTSSTGSP.
  • the insert was obtained by annealing the following pairs of phosphorylated synthetic oligonucleotide pairs:
  • XTEN_AE864 was constructed from serial dimerization of XTEN AE36 to AE72, 144, 288, 576 and 864.
  • a collection of XTEN_AE72 segments was constructed from 37 different segments of XTEN AE36.
  • Cultures of E. coli harboring all 37 different 36-amino acid segments were mixed and plasmid was isolated. This plasmid pool was digested with Bsal/Ncol to generate the small fragment as the insert. The same plasmid pool was digested with Bbsl/Ncol to generate the large fragment as the vector.
  • the insert and vector fragments were ligated resulting in a doubling of the length and the ligation mixture was transformed into BL21Gold(DE3) cells to obtain colonies of XTEN AE72.
  • This library of XTEN AE72 segments was designated LCW0406. All clones from LCW0406 were combined and dimerized again using the same process as described above yielding library LCW0410 of XTEN_AE144. All clones from LCW0410 were combined and dimerized again using the same process as described above yielding library LCW0414 of XTEN AE288. Two isolates
  • LCW0414.001 and LCW0414.002 were randomly picked from the library and sequenced to verify the WSGR Dkt Ref. 32808-726601 identities. All clones from LCW0414 were combined and dimerized again using the same process as described above yielding library LCW0418 of XTEN_AE576.
  • a collection of XTEN AM144 segments was constructed starting from 37 different segments of XTEN_AE36, 44 segments of XTEN AF36, and 44 segments of XTEN AG36.
  • LCW0462 1512 Isolates from library LCW0462 were screened for protein expression. Individual colonies were transferred into 96 well plates and cultured overnight as starter cultures. These starter cultures were diluted into fresh autoinduction medium and cultured for 20-30h. Expression was measured using a fluorescence plate reader with excitation at 395 nm and emission at 510 nm. 192 isolates showed high level expression and were submitted to DNA sequencing. Most clones in library LCW0462 showed good expression and similar physicochemical properties suggesting that most combinations of
  • XTEN_AM36 segments yield useful XTEN sequences.
  • 30 isolates from LCW0462 were chosen as a preferred collection of XTEN AM144 segments for the construction of multifunctional proteins that contain multiple XTEN segments.
  • the file names of the nucleotide and amino acid constructs for these segments are listed in Table 13.
  • Example 8 Construction of XTEN_AM432
  • the stuffer vector pCW0359 was digested with Bsal and pnl to remove the stuffer segment and the resulting vector fragment was isolated by agarose gel purification.
  • CCTCGAGTGAAGACGAACCTCCCGTGCTTGGCGCGCCGCTTGCGCTTGC for introducing the sequencing island A (SI-A) which encodes amino acids GASASGAPSTG and has the restriction enzyme Ascl recognition nucleotide sequence GGCGCGCC inside.
  • SI-A sequencing island A
  • the annealed oligonucleotide pairs were ligated with Bsal and pnl digested stuffer vector pCW0359 prepared above to yield pCW0466 containing SI-A.
  • pCW0466 containing SI-A.
  • We then generated a library of XTEN_AM443 segments by recombining 43 preferred XTEN AM432 segments from Example 8 and SI-A segments from pCW0466 at C-terminus using the same dimerization process described in Example 5. This new library of XTEN_AM443 segments was designated LCW0479.
  • CCTCGAGTGAAGACGAACCTCCGCTTGGGGCCGGCCCCGTTGGTTCTGG for introducing the sequencing island B (SI-B) which encodes amino acids GPEPTGPAPSG and has the restriction enzyme Fsel recognition nucleotide sequence GGCCGGCC inside.
  • the annealed oligonucleotide pairs were ligated with Bsal and Kpnl digested stuffer vector pCW0359 as used in Example 9 to yield pCW0467 containing SI-B.
  • We then generated a library of XTEN_AM443 segments by recombining 43 preferred XTEN_AM432 segments from Example 8 and SI-B segments from pCW0467 at C-terminus using the same dimerization process described in Example 5. This new library of XTEN AM443 segments was designated LCW0480.
  • XTEN AD864 sequences starting from segments of XTEN AD36 listed in Example 1. These sequences were assembled as described in Example 5. Several isolates from XTEN_AD864 were evaluated and found to show good expression and excellent solubility under physiological conditions. One intermediate WSGR Dkt Ref. 32808-726601 construct of XTEN AD576 was sequenced. This clone was evaluated in a PK experiment in cynomolgus monkeys and a half-life of about 20h was measured.
  • XTEN AF864 sequences starting from segments of XTEN AF36 listed in Example 3. These sequences were assembled as described in Example 5. Several isolates from XTEN AP864 were evaluated and found to show good expression and excellent solubility under physiological conditions.
  • One intermediate construct of XTEN AF540 was sequenced. This clone was evaluated in a PK experiment in cynomolgus monkeys and a half-life of about 20h was measured. A full length clone of XTEN AF864 had excellent solubility and showed half-life exceeding 60h in cynomolgus monkeys.
  • a second set of XTEN AF sequences was assembled including a sequencing island as described in Example 9.
  • XTEN AG864 sequences starting from segments of XTEN_AD36 listed in Example 1. These sequences were assembled as described in Example 5. Several isolates from XTEN AG864 were evaluated and found to show good expression and excellent solubility under physiological conditions. A full-length clone of XTEN_AG864 had excellent solubility and showed half-life exceeding 60h in cynomolgus monkeys.
  • This example details a step in the optimization of the N-terminus of the XTEN protein to promote the initiation of translation to allow for expression of XTEN fusions at the N-terminus of fusion proteins without the presence of a helper domain.
  • Historically expression of proteins with XTEN at the N-terminus was poor, yielding values that would essentially undetectable in the GFP fluorescence assay ( ⁇ 25% of the expression with the N-terminal CBD helper domain).
  • To create diversity at the codon level seven amino acid sequences were selected and prepared with a diversity of codons.
  • Table 14 Theoretical Diversity and Sampling Numbers for 12mer Addition Libraries. The amino acid residues with randomized codons are underlined. WSGR Dkt Ref. 32808-726601
  • Example IS Construction of N-terminal extensions of XTEN- Construction and screening of Libraries Optimizing codons 3 and 4
  • This example details a step in the optimization of the N-terminus of the XTEN protein to promote the initiation of translation to allow for expression of XTEN fusions at the N-terminus of proteins without the presence of a helper domain.
  • the third and fourth codons were randomized to determine preferences.
  • This example details a step in the optimization of the N-terminus of the XTEN protein to promote the initiation of translation to allow for expression of XTEN fusions at the N-terminus of proteins without the presence of a helper domain.
  • the N-terminus was examined in a broader context by combining the 12 selected 12mer sequences (see Example supra) at the very N-terminus followed by 125 previously constructed 36mer segments (see example supra) in a combinatorial manner. This created novel 48mers at the N- terminus of the XTEN protein and enabled the assessment of the impact of longer-range interactions at the N-terminus on expression of the longer sequences (FIG. 29).
  • the plasmids containing the 125 selected 36mer segments were digested with restriction enzymes Bbsl Ncol and the appropriate fragment was gel-purified.
  • the plasmid from clone AC94 CBD-XTEN AM875-GFP was also digested with Bsal/Ncol and the appropriate fragments were gel-purified. These fragments were ligated together and transformed into E. coli BL21Gold(DE3) competent cells to obtain colonies of the library LCW0579, which also served as the vector for further cloning 12 selected 12mers at the very N-terminus.
  • the plasmids of LCW0579 were digested with Ndel EcoRI Bsal and the appropriate fragments were gel-purified. 12 pairs of
  • oligonucleotides encoding 12 selected 12mer sequences were designed, annealed and ligated with the Ndel EcoRI Bsal digested LCW0579 vector, and transformed into E. coli BL21Gold(DE3) competent cells to obtain colonies of the library LCW0580. With a theoretical diversity of 1500 unique clones, a total of 1512 individual colonies from the created library were picked and grown overnight to saturation in 500 ⁇ of super broth media in a 96 deep well plate. This provided sufficient coverage to understand relative library performance and sequence preferences. The saturated overnight cultures were used to inoculate new 500 ⁇ cultures in auto-induction media that were grown overnight at 26°C.
  • This example details a step in the optimization of the N-terminus of the XTEN protein to promote the initiation of translation to allow for expression of XTEN fusions at the N-terminus of proteins without the presence of a helper domain.
  • a combinatorial approach was undertaken to examine the union of these preferences. This created novel 48mers at the N-terminus of the XTEN protein and enabled the testing of the confluence of previous conclusions. Additionally, the ability of these leader sequences to be a universal solution for all XTEN proteins was assessed by placing the new 48mers in front of both XTEN-AE864 and XTEN- AM875.
  • the plasmids from 6 selected clones of 36mer segment with best GFP expression in the combinatorial library were digested with Ndel/EcoRI/Bsal and the appropriate fragments were gel-purified.
  • the plasmids from clones AC94 CBD-XTEN AM875- WSGR Dkt Ref. 32808-726601
  • GFP and AC 104 were digested with digested with Ndel EcoRI/Bsal and the appropriate fragments were gel-purified. These fragments were ligated together and transformed into E. coli BL21Gold(DE3) competent cells to obtain colonies of the libraries LCW0585 (-XTEN_AM875- GFP) and LCW0586 (-XTEN_AE864-GFP), which could also serve as the vectors for further cloning 8 selected 12mers at the very N-terminus.
  • the plasmids of LCW0585 and LCW0586 were digested with Ndel EcoRI/Bsal and the appropriate fragments were gel-purified.
  • the saturated overnight cultures were used to inoculate new 500 ⁇ cultures in auto-induction media in which were grown overnight at 26°C. These expression cultures were then assayed using a fluorescence plate reader (excitation 395 nm, emission 510 nm) to determine the amount of GFP reporter present.
  • the top 36 clones were sequenced and retested for GFP reporter expression. 36 clones yielded usable sequencing data and these 36 were used for the subsequent analysis.
  • the sequencing data determined the 12mer, the third codon, the fourth codon and the 36mer present in the clone and revealed that many of the clones were independent replicates of the same sequence. Additionally, the retest results for these clones are close in value, indicating the screening process was robust.
  • the preferred combination of the N-terminal for the XTEN-AM875 and the preferred combination for the XTEN-AE864 are not the same, indicating more complex interactions further than 150 bases from the initiation site influence expression levels.
  • the sequences for the preferred nucleotide sequences are listed in Table 22 and the preferred clones were analyzed by SDS-PAGE to independently confirm expression (see FIG. 30). The complete sequences of XTEN AM923 and XTEN AE912 were selected for further analysis.
  • Table 22 Preferred DNA Nucleotide Sequences for first 48 Amino Acid Residues of N-terminal XTEN-AM875 and XTEN-AE864
  • Example 18 Methods of producing and evaluating CFXTEN; XTEN-CF as example
  • FIG. 33 A general schema for producing and evaluating CFXTEN compositions is presented in FIG. 33, and forms the basis for the general description of this Example.
  • a skilled artesian can create and evaluate a range of CFXTEN fusion proteins comprising, XTENs, CF and variants of CF known in the art.
  • the Example is, therefore, to be construed as merely illustrative, and not limitative of the methods in any way whatsoever; numerous variations will be apparent to the ordinarily skilled artisan.
  • a CFXTEN of coagulation factor linked to an XTEN of the AE family of motifs is created.
  • FIG. 32 is a schematic flowchart of representative steps in the assembly of a XTEN
  • sequence motifs 502 such as a 12 amino acid motif ("12-mer"), which is subsequently WSGR Dkt Ref. 32808-726601 ligated with an oligo containing Bbsl, and Kpnl restriction sites 503.
  • the motif libraries can be limited to specific sequence XTEN families; e.g., AD, AE, AF, AG, AM, or AQ sequences of Table 3.
  • the motifs of the AE family are used as the motif library, which are annealed to the 12-mer to create a "building block" length; e.g., a segment that encodes 36 amino acids.
  • the gene encoding the XTEN sequence can be assembled by ligation and multimerization of the "building blocks” until the desired length of the XTEN gene 504 is achieved.
  • the XTEN length in this case is 48 amino acid residues, but longer lengths can be achieved by this process.
  • multimerization can be performed by ligation, overlap extension, PCR assembly or similar cloning techniques known in the art.
  • the XTEN gene can be cloned into a stuffer vector. In the example illustrated in FIG.
  • the vector can encode a Flag sequence 506 followed by a stuffer sequence that is flanked by Bsal, Bbsl, and Kpnl sites 507 and a CF gene (e.g., FVII) 508, resulting in the gene encoding the CFXTEN 500, which, in this case encodes the fusion protein in the configuration, N- to C-terminus, XTEN-FVII.
  • FVII CF gene
  • DNA sequences encoding CF can be conveniently obtained by standard procedures known in the art from a cDNA library prepared from an appropriate cellular source, from a genomic library, or may be created synthetically (e.g., automated nucleic acid synthesis) using DNA sequences obtained from publicly available databases, patents, or literature references.
  • a gene or polynucleotide encoding the CF portion of the protein or its complement can be then be cloned into a construct, such as those described herein, which can be a plasmid or other vector under control of appropriate transcription and translation sequences for high level protein expression in a biological system.
  • a second gene or polynucleotide coding for the XTEN portion or its complement in the case of FIG.
  • XTEN 32 illustrated as an XTEN with 48 amino acid residues can be genetically fused to the nucleotides encoding the terminus of the CF gene by cloning it into the construct adjacent and in frame with the gene coding for the CF, through a ligation or multimerization step. In this manner, a chimeric DNA molecule coding for (or complementary to) the CFXTEN fusion protein Re generated within the construct.
  • a gene encoding for a second XTEN are inserted and ligated in-frame to the nucleotides encoding the opposite terminus of the CFXTEN gene or can be inserted within the CF-encoding region.
  • the construct can be designed in different configurations to encode the various permutations of the fusion partners as a monomeric polypeptide.
  • the gene can be created to encode the fusion protein in the order (N- to C- terminus): CF-XTEN; XTEN-CF; CF-XTEN- CF; XTEN- CF-XTEN; as well as multimers of the foregoing.
  • this chimeric DNA molecule is transferred or cloned into another construct that is a more appropriate expression vector.
  • a host cell capable of expressing the chimeric DNA molecule is transformed with the chimeric DNA molecule.
  • the vectors containing the DNA segments of interest can be transferred into an appropriate host cell by well-known methods, depending on the type of cellular host, as described supra. WSGR Dkt Ref. 32808-726601
  • Host cells containing the XTEN-CF expression vector is cultured in conventional nutrient media modified as appropriate for activating the promoter.
  • the culture conditions such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
  • culture broth is harvested and separated from the cell mass and the resulting crude extract retained for purification of the fusion protein.
  • Gene expression are measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA [Thomas, Proc. Natl. Acad. Sci. USA, 77:5201 -5205 (1980)], dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein.
  • gene expression is measured by immunological of fluorescent methods, such as immunohistochemical staining of cells to quantitate directly the expression of gene product.
  • Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal,, and may be prepared in any mammal.
  • the antibodies may be prepared against the CF sequence polypeptide using a synthetic peptide based on the sequences provided herein or against exogenous sequence fused to CF and encoding a specific antibody epitope.
  • selectable markers are well known to one of skill in the art and include reporters such as enhanced green fluorescent protein (EGFP), beta-galactosidase ( ⁇ -gal) or chloramphenicol acetyltransferase (CAT).
  • the CFXTEN polypeptide product is purified via methods known in the art. Procedures such as gel filtration, affinity purification, salt fractionation, ion exchange chromatography, size exclusion chromatography, hydroxyapatite adsorption chromatography, hydrophobic interaction chromatography or gel electrophoresis are all techniques that may be used in the purification. Specific methods of purification are described in Robert . Scopes, Protein Purification: Principles and Practice, Charles R. Castor, ed., Springer-Verlag 1994, and Sambrook, et ah, supra. Multi-step purification separations are also described in Baron, et al., Crit. Rev. Biotechnol. 10: 179-90 (1990) and Below, et al., J. Chromatogr. A. 679:67-83 (1994).
  • the isolated CFXTEN fusion proteins would then be characterized for their chemical and activity properties.
  • Isolated fusion protein is characterized, e.g., for sequence, purity, apparent molecular weight, solubility and stability using standard methods known in the art.
  • the fusion protein meeting expected standards would then be evaluated for activity, which can be measured in vitro or in vivo by measuring one of the coagulation factor-associated parameters described herein, using one or more assays disclosed herein, or using the assays of the Examples or Table 40.
  • the XTEN-CF fusion protein is administered to one or more animal species to determine standard pharmacokinetic parameters and pharmacodynamic properties, as described in Examples 30-33.
  • the CFXTEN compositions comprising CF and an XTEN can be produced and evaluated by one of ordinary WSGR Dkt Ref. 32808-726601 skill in the art to confirm the expected properties such as enhanced solubility, enhanced stability, improved pharmacokinetics and reduced immunogenicity, leading to an overall enhanced therapeutic activity compared to the corresponding unfused CF.
  • a different sequence can be constructed, expressed, isolated and evaluated by these methods in order to obtain a composition with such properties.
  • Example 19 Construction of expression plasmids for FVII-XTEN
  • the cloning vector containing the gene encoding FVII was purchased from OriGene
  • Transformants were screened by DNA miniprep and the desired constructs were confirmed by DNA sequencing.
  • the final construct is pCW0647.001 which encodes the FVII-TEV-XTEN AE864 protein
  • FVII was amplified with pCW0647.001 as a template.
  • the PCR primers introduced Nhel and Bsal restriction enzyme recognition sequences on the 5' and 3' end respectively and deleted the TEV site.
  • the Nhel/Bsal digested FVII fragment was fused to Bsal/Hindlll digested XTEN AE864 fragment and inserted into Nhel/Hindlll digested pSecTag2C expression vector.
  • the ligated DNA mixture was electroporated into XL 1 -Blue bacterial cells. Transformants were screened by DNA miniprep and the desired constructs were confirmed by DNA sequencing.
  • the final construct is pCW0645.001 which encodes the FVII-XTEN AE864 protein (Table 23).
  • Expression vector pCW0645.001 was digested with Nhel and Sail. The resulting 4091 bp fragment included nucleotides that encode the FVII-XTEN_AE864 protein. This fragment was ligated with Nhel Sall digested CET1019-AS-puro, CET1019-HS-puro, SC AS-puro, or DC HS-puro (licensed from Millipore). These vectors feature a CMV promoter that lies upstream of the gene insertion site, and the CET1019 vectors also contain a UCOE element upstream of the promoter. The ligated DNA mixture was electroporated into XLl-Blue bacterial cells.
  • Transformants were screened by DNA miniprep and the desired constructs were confirmed by DNA sequencing.
  • the resulting expression vectors were AC397 (pBC0013, SC AS puro-FVD-XTEN_AE864), AC402 (pBC0014, SC HS puro-FVII- XTEN_AE864), AC403 (pBC0015, CET1019 AS puro-FVII-XTEN_AE864), and AC404 (pBC0016, CET1019 HS puro-FVII-XTEN_AE864)
  • Expression vector pCW0645.001 was digested with Bsal and Hindlll. The resulting 6400bp fragment was ligated with Bsal Hindlll digested XTEN_AE288 fragment. The ligated DNA mixture was electroporated into XL 1 -Blue bacterial cells. Transformants were screened by DNA miniprep and the desired constructs were confirmed by DNA sequencing. The resulting expression vector was pBC0019 ( P SecTag2C-FVn-XTEN_AE288).
  • Expression vector pBC0019 was digested with Nhel and Sail. The resulting 2363bp fragment included nucleotides that encode the FVU-XTEN AE288 protein. This fragment was ligated with Nhel/Sall digested CET1019-AS-puro, or CET1019-HS-puro (licensed from Millipore). These vectors feature a CMV promoter and a UCOE element that lie upstream of the gene insertion site. The ligated DNA mixture was electroporated into XL 1 -Blue bacterial cells. Transformants were screened by DNA miniprep and the desired constructs were confirmed by DNA sequencing.
  • the resulting expression vectors were AC405 (pBC0017, CET1019 AS puro- FVII-XTEN AE288), and AC398 (pBC0018, CET1019 HS puro-FVn-XTEN_AE288) (Table 23).
  • RNASKPQGRIVGGKVC GCAGTACTGCAGTGACCACACGGGCACCAAGCGCTCC
  • VAQVIIPSTYVPGTTNH AGGGGAGTGTCCATGGCAGGTCCTGTTGTTGGTGAAT
  • GATALELMVLNVPRLM ACGACCTCAGCGAGCACGACGGGGATGAGCAGAGCCG
  • TQDCLQQSRKVGDSPNI GCGGGTGGCGCAGGTCATCATCCCCAGCACGTACGTC
  • TEYMFCAGYSDGSKDS CCGGGCACCACCAACCACGACATCGCGCTGCTCCGCC
  • APGSPAGSPTSTEEGTS AGCAAGGACTCCTGCAAGGGGGACAGTGGAGGCCCAC
  • SAPGTSESATPESGPGS CATCGTCAGCTGGGGCCAGGGCTGCGCAACCGTGGGC

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Abstract

La présente invention porte sur des compositions comprenant des facteurs de coagulation DC liés à un polypeptide recombinant étendu (XTEN), sur des acides nucléiques isolés codant pour les compositions et sur des vecteurs et cellules hôtes les contenant, et sur des procédés de fabrication et d'utilisation de telles compositions dans le traitement de maladies, de troubles et d'états liés à un facteur de coagulation.
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Families Citing this family (75)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7855279B2 (en) 2005-09-27 2010-12-21 Amunix Operating, Inc. Unstructured recombinant polymers and uses thereof
CN102348715B (zh) 2009-02-03 2017-12-08 阿穆尼克斯运营公司 延伸重组多肽和包含该延伸重组多肽的组合物
US8703717B2 (en) 2009-02-03 2014-04-22 Amunix Operating Inc. Growth hormone polypeptides and methods of making and using same
NZ596778A (en) 2009-06-08 2013-11-29 Amunix Operating Inc Glucose-regulating polypeptides and methods of making and using same
US9849188B2 (en) 2009-06-08 2017-12-26 Amunix Operating Inc. Growth hormone polypeptides and methods of making and using same
AU2010290077C1 (en) 2009-08-24 2015-12-03 Bioverativ Therapeutics Inc. Coagulation factor IX compositions and methods of making and using same
WO2011123830A2 (fr) * 2010-04-02 2011-10-06 Amunix Operating Inc. Compositions d'alpha 1-antitrypsine, leurs procédés de préparation et d'utilisation
SI2717898T1 (sl) 2011-06-10 2019-07-31 Bioverativ Therapeutics Inc. Spojine prokoagulantov in metode za njihovo uporabo
US10656167B2 (en) 2011-07-25 2020-05-19 Bioverativ Therapeutics Inc. Assays to monitor bleeding disorders
EP3431098A1 (fr) * 2011-09-12 2019-01-23 Amunix Operating Inc. Compositions de peptide-2 de type glucagon et leurs procédés de fabrication et d'utilisation
WO2013106787A1 (fr) 2012-01-12 2013-07-18 Biogen Idec Ma Inc. Facteur viii chimérique et polypeptides et leurs utilisations
NZ628014A (en) 2012-02-15 2016-09-30 Biogen Ma Inc Recombinant factor viii proteins
RS63870B1 (sr) 2012-02-15 2023-01-31 Bioverativ Therapeutics Inc Sastavi faktora viii i postupci za pravljenje i upotrebu istih
WO2013130684A1 (fr) 2012-02-27 2013-09-06 Amunix Operating Inc. Compositions de conjugués xten-folate et des procédés de préparation de celles-ci
WO2013162078A1 (fr) * 2012-04-27 2013-10-31 学校法人日本大学 Agent thérapeutique pour une lésion dans un épithélium et un endothélium
AU2013270682A1 (en) 2012-06-08 2014-12-11 Biogen Ma Inc. Procoagulant compounds
EP2863940A4 (fr) 2012-06-08 2016-08-10 Biogen Ma Inc Facteurs de coagulation chimériques
KR102403545B1 (ko) 2012-07-11 2022-05-30 바이오버라티브 테라퓨틱스 인크. Xten 및 폰 빌레브란트 인자 단백질과의 viii 인자 복합체 및 이의 용도
CN102876695A (zh) * 2012-09-12 2013-01-16 天津工业生物技术研究所 一种含有Z-Tag蛋白融合标签的快速克隆及表达质粒载体
WO2014100913A1 (fr) * 2012-12-24 2014-07-03 Beijing Anxinhuaide Biotech. Co., Ltd Amélioration de la demi-vie d'un polypeptide thérapeutique par fusion avec une protéine d'échafaudage trimère via un espaceur
ES2959747T3 (es) 2013-02-15 2024-02-28 Bioverativ Therapeutics Inc Gen del factor VIII optimizado
SG11201505926VA (en) 2013-03-15 2015-09-29 Biogen Ma Inc Factor ix polypeptide formulations
SG10201913738YA (en) 2013-06-28 2020-03-30 Bioverativ Therapeutics Inc Thrombin cleavable linker with xten and its uses thereof
WO2015021423A2 (fr) 2013-08-08 2015-02-12 Biogen Idec Ma Inc. Purification de molécules chimères du facteur viii
EP3033097B1 (fr) 2013-08-14 2021-03-10 Bioverativ Therapeutics Inc. Fusions de factor viii-xten et leurs utilisations.
US20160311885A1 (en) 2013-08-14 2016-10-27 Biogen Ma Inc. Recombinant factor viii proteins
HUE057005T2 (hu) * 2013-09-25 2022-04-28 Bioverativ Therapeutics Inc Oszlopon történõ vírusinaktiváló eljárások
WO2015070014A1 (fr) 2013-11-08 2015-05-14 Biogen Idec Ma Inc. Composé de fusion procoagulant
CN117106095A (zh) 2014-01-10 2023-11-24 比奥贝拉蒂治疗公司 因子viii嵌合蛋白及其用途
EP3102589A1 (fr) 2014-02-04 2016-12-14 Biogen MA Inc. Chromatographie par échange cationique utilisée dans le mode de fixation par écoulement pour enrichir les modifications post-traductionnelles
WO2015154090A1 (fr) * 2014-04-04 2015-10-08 Bloodworks Laboratoire ordinaire et analyse hors laboratoire de l'hémostase
CN105294857B (zh) * 2014-06-18 2019-02-19 上海交通大学 基于fix的抗原表位及其应用
WO2016004113A1 (fr) 2014-06-30 2016-01-07 Biogen Ma Inc. Gène du facteur ix optimisé
US10676731B2 (en) 2014-08-19 2020-06-09 The Children's Hospital Of Philadelphia Compositions and methods for modulating factor IX function
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MA40835A (fr) 2014-10-23 2017-08-29 Biogen Ma Inc Anticorps anti-gpiib/iiia et leurs utilisations
MA40864A (fr) 2014-10-31 2017-09-05 Biogen Ma Inc Hypotaurine, gaba, bêta-alanine et choline pour la régulation de l'accumulation de sous-produits résiduaires dans des procédés de culture de cellules mammifères
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TWI741992B (zh) * 2015-08-03 2021-10-11 美商百歐維拉提夫治療公司 因子ｉｘ融合蛋白以及其製備及使用方法
CA2992306A1 (fr) 2015-08-28 2017-03-09 Amunix Operating Inc. Ensemble de polypeptides chimeriques et procedes de preparation et d'utilisation de ceux-ci
CN105524147B (zh) * 2015-10-15 2020-06-09 中国药科大学 重组聚多肽及其应用
AU2016362606A1 (en) * 2015-12-03 2018-06-28 Takeda Pharmaceutical Company Limited Factor VIII with extended half-life and reduced ligand-binding properties
JP6844890B2 (ja) * 2016-01-07 2021-03-17 藤森工業株式会社 採血管、試薬及びそれらを利用した血液性状分析方法
HRP20221089T1 (hr) 2016-02-01 2022-11-25 Bioverativ Therapeutics Inc. Optimizirani geni faktora viii
US10738338B2 (en) 2016-10-18 2020-08-11 The Research Foundation for the State University Method and composition for biocatalytic protein-oligonucleotide conjugation and protein-oligonucleotide conjugate
EP3548066A1 (fr) 2016-12-02 2019-10-09 Bioverativ Therapeutics Inc. Méthodes de traitement de l'arthropathie hémophilique à l'aide de facteurs de coagulation chimériques
CN110520149A (zh) 2016-12-02 2019-11-29 比奥维拉迪维治疗股份有限公司 诱导对凝血因子的免疫耐受性的方法
US11180535B1 (en) 2016-12-07 2021-11-23 David Gordon Bermudes Saccharide binding, tumor penetration, and cytotoxic antitumor chimeric peptides from therapeutic bacteria
US11129906B1 (en) 2016-12-07 2021-09-28 David Gordon Bermudes Chimeric protein toxins for expression by therapeutic bacteria
US20200031895A1 (en) 2016-12-16 2020-01-30 Biogen Ma Inc. Stabilized proteolytically activated growth differentiation factor 11
AU2018215092A1 (en) 2017-01-31 2019-08-29 Bioverativ Therapeutics Inc. Factor IX fusion proteins and methods of making and using same
CA3051639A1 (fr) 2017-02-01 2018-08-09 Novo Nordisk A/S Anticorps procoagulants
EP3612637A4 (fr) 2017-04-21 2021-01-27 Yuhan Corporation Procédé de production de protéines à double fonction et ses dérivés
US20210163986A1 (en) 2017-08-09 2021-06-03 Bioverativ Therapeutics Inc. Nucleic acid molecules and uses thereof
US20200352996A1 (en) * 2017-09-27 2020-11-12 Ohio State Innovation Foundation Tissue factor-targeting car-nk and car-t cell therapy
AU2018393111A1 (en) 2017-12-21 2020-07-23 Amunix Pharmaceuticals, Inc. Release segments and binding compositions comprising same
AU2019215063A1 (en) 2018-02-01 2020-09-03 Bioverativ Therapeutics, Inc. Use of lentiviral vectors expressing Factor VIII
CA3094112A1 (fr) 2018-03-28 2019-10-03 Bristol-Myers Squibb Company Proteines de fusion de l'interleukine-2/du recepteur alpha de l'interleukine-2 et procedes d'utilisation
JP2021523878A (ja) 2018-05-18 2021-09-09 バイオベラティブ セラピューティクス インコーポレイテッド 血友病ａを処置する方法
BR112020026512A2 (pt) 2018-07-03 2021-04-06 Bristol-Myers Squibb Company Formulações de fgf-21
US20200069817A1 (en) 2018-08-09 2020-03-05 Bioverativ Therapeutics Inc. Nucleic acid molecules and uses thereof for non-viral gene therapy
BR112021008105A2 (pt) 2018-10-29 2021-08-03 Biogen Ma Inc. variantes de fc5 humanizadas e estabilizadas para intensificação de transporte através de barreira hematoencefálica
CN113396223A (zh) 2018-12-06 2021-09-14 比奥维拉迪维治疗股份有限公司 表达因子ix的慢病毒载体的用途
JP2022531095A (ja) 2019-04-17 2022-07-06 コディアック バイオサイエンシーズ， インコーポレイテッド エキソソーム及びａａｖの組成物
CA3143584A1 (fr) 2019-06-18 2020-12-24 Bayer Aktiengesellschaft Analogues d'adrenomedulline pour stabilisation a long terme et leur utilisation
WO2021067389A1 (fr) 2019-09-30 2021-04-08 Bioverativ Therapeutics Inc. Formulations de vecteur lentiviral
EP4058466A1 (fr) 2019-11-13 2022-09-21 Amunix Pharmaceuticals, Inc. Polypeptides xten à code à barres, compositions associées et leurs procédés de production et d'utilisation
IL299333A (en) 2020-06-24 2023-02-01 Bioverativ Therapeutics Inc Methods for the purification of viral vectors
AU2022334711A1 (en) 2021-08-23 2024-04-04 Bioverativ Therapeutics Inc. Baculovirus expression system
KR20240049332A (ko) 2021-08-23 2024-04-16 바이오버라티브 테라퓨틱스 인크. 최적화된 인자 viii 유전자
KR20240067122A (ko) 2021-09-30 2024-05-16 바이오버라티브 테라퓨틱스 인크. 감소된 면역원성을 갖는 인자 viii 폴리펩타이드를 암호화하는 핵산
EP4342460A1 (fr) 2022-09-21 2024-03-27 NovoArc GmbH Nanoparticule lipidique avec charge d'acide nucléique
US20240327538A1 (en) 2023-02-10 2024-10-03 Amunix Pharmaceuticals, Inc. Compositions targeting prostate-specific membrane antigen and methods for making and using the same

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007103515A2 (fr) * 2006-03-06 2007-09-13 Amunix, Inc. Polymères recombinés non structurés et utilisations correspondantes
WO2008155134A1 (fr) * 2007-06-21 2008-12-24 Technische Universität München Protéines biologiquement actives présentant une stabilité in vivo et/ou in vitro accrue

Family Cites Families (352)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4179337A (en) 1973-07-20 1979-12-18 Davis Frank F Non-immunogenic polypeptides
US3992518A (en) 1974-10-24 1976-11-16 G. D. Searle & Co. Method for making a microsealed delivery device
GB1478759A (en) 1974-11-18 1977-07-06 Alza Corp Process for forming outlet passageways in pills using a laser
US4284444A (en) 1977-08-01 1981-08-18 Herculite Protective Fabrics Corporation Activated polymer materials and process for making same
US4200098A (en) 1978-10-23 1980-04-29 Alza Corporation Osmotic system with distribution zone for dispensing beneficial agent
US4200984A (en) 1979-03-12 1980-05-06 Fink Ray D Detachable tool combining bracket and method
US4342832A (en) 1979-07-05 1982-08-03 Genentech, Inc. Method of constructing a replicable cloning vehicle having quasi-synthetic genes
US4215051A (en) 1979-08-29 1980-07-29 Standard Oil Company (Indiana) Formation, purification and recovery of phthalic anhydride
ZA811368B (en) 1980-03-24 1982-04-28 Genentech Inc Bacterial polypedtide expression employing tryptophan promoter-operator
US4398908A (en) 1980-11-28 1983-08-16 Siposs George G Insulin delivery system
US4456591A (en) 1981-06-25 1984-06-26 Baxter Travenol Laboratories, Inc. Therapeutic method for activating factor VII
US4435173A (en) 1982-03-05 1984-03-06 Delta Medical Industries Variable rate syringe pump for insulin delivery
US4542025A (en) 1982-07-29 1985-09-17 The Stolle Research And Development Corporation Injectable, long-acting microparticle formulation for the delivery of anti-inflammatory agents
US4599311A (en) 1982-08-13 1986-07-08 Kawasaki Glenn H Glycolytic promotersfor regulated protein expression: protease inhibitor
US4713339A (en) 1983-01-19 1987-12-15 Genentech, Inc. Polycistronic expression vector construction
US4870008A (en) 1983-08-12 1989-09-26 Chiron Corporation Secretory expression in eukaryotes
US4757006A (en) 1983-10-28 1988-07-12 Genetics Institute, Inc. Human factor VIII:C gene and recombinant methods for production
US5004804A (en) 1984-01-12 1991-04-02 Nordisk Gentofte Method and composition for preparation of factor VIIIC
US7138505B1 (en) 1984-01-12 2006-11-21 Novartis Vaccines And Diagnostics, Inc. Factor VIII:C nucleic acid molecules
DE3572982D1 (en) 1984-03-06 1989-10-19 Takeda Chemical Industries Ltd Chemically modified lymphokine and production thereof
US5231112A (en) 1984-04-12 1993-07-27 The Liposome Company, Inc. Compositions containing tris salt of cholesterol hemisuccinate and antifungal
US5916588A (en) 1984-04-12 1999-06-29 The Liposome Company, Inc. Peptide-containing liposomes, immunogenic liposomes and methods of preparation and use
US4965199A (en) 1984-04-20 1990-10-23 Genentech, Inc. Preparation of functional human factor VIII in mammalian cells using methotrexate based selection
US4931373A (en) 1984-05-25 1990-06-05 Zymogenetics, Inc. Stable DNA constructs for expression of α-1 antitrypsin
JPS61502760A (ja) 1984-07-24 1986-11-27 キイ・フア−マシユ−テイカルズ・インコ−ポレイテツド 粘着性経皮投与層
US4766073A (en) 1985-02-25 1988-08-23 Zymogenetics Inc. Expression of biologically active PDGF analogs in eucaryotic cells
US4885249A (en) 1984-12-05 1989-12-05 Allelix, Inc. Aspergillus niger transformation system
US4965188A (en) 1986-08-22 1990-10-23 Cetus Corporation Process for amplifying, detecting, and/or cloning nucleic acid sequences using a thermostable enzyme
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US5981216A (en) 1985-04-01 1999-11-09 Alusuisse Holdings A.G. Transformed myeloma cell-line and a process for the expression of a gene coding for a eukaryotic polypeptide employing same
CA1341174C (fr) 1985-04-12 2001-01-30 John J. Toole Jr. Proteines procoagulantes derivees du facteur viii: c
GR860984B (en) 1985-04-17 1986-08-18 Zymogenetics Inc Expression of factor vii and ix activities in mammalian cells
ATE74164T1 (de) 1985-04-22 1992-04-15 Genetics Inst Herstellung mit hoher leistung des aktivfaktors ix.
US4684479A (en) 1985-08-14 1987-08-04 Arrigo Joseph S D Surfactant mixtures, stable gas-in-liquid emulsions, and methods for the production of such emulsions from said mixtures
KR910006424B1 (ko) 1985-08-21 1991-08-24 인코텍스 비.브이 편성브리프(brief) 제조방법
US4882279A (en) 1985-10-25 1989-11-21 Phillips Petroleum Company Site selective genomic modification of yeast of the genus pichia
US5250421A (en) 1986-01-03 1993-10-05 Genetics Institute, Inc. Method for producing factor VIII:C-type proteins
DE3785102T2 (de) 1986-01-03 1993-07-22 Genetics Inst Verfahren zur herstellung von faktor-viii:c-typ-proteinen.
US5198349A (en) 1986-01-03 1993-03-30 Genetics Institute, Inc. Method for producing factor VIII:C and analogs
US4935349A (en) 1986-01-17 1990-06-19 Zymogenetics, Inc. Expression of higher eucaryotic genes in aspergillus
US5595886A (en) 1986-01-27 1997-01-21 Chiron Corporation Protein complexes having Factor VIII:C activity and production thereof
US4800159A (en) 1986-02-07 1989-01-24 Cetus Corporation Process for amplifying, detecting, and/or cloning nucleic acid sequences
DK122686D0 (da) 1986-03-17 1986-03-17 Novo Industri As Fremstilling af proteiner
GB8610600D0 (en) 1986-04-30 1986-06-04 Novo Industri As Transformation of trichoderma
US6759057B1 (en) 1986-06-12 2004-07-06 The Liposome Company, Inc. Methods and compositions using liposome-encapsulated non-steroidal anti-inflammatory drugs
US5543502A (en) 1986-06-24 1996-08-06 Novo Nordisk A/S Process for producing a coagulation active complex of factor VIII fragments
IE60901B1 (en) 1986-08-21 1994-08-24 Vestar Inc Improved treatment of systemic fungal infections with phospholipid particles encapsulating polyene antifungal antibiotics
US4933185A (en) 1986-09-24 1990-06-12 Massachusetts Institute Of Technology System for controlled release of biologically active compounds
US6024983A (en) 1986-10-24 2000-02-15 Southern Research Institute Composition for delivering bioactive agents for immune response and its preparation
US4912040A (en) 1986-11-14 1990-03-27 Genetics Institute, Inc. Eucaryotic expression system
US6406713B1 (en) 1987-03-05 2002-06-18 The Liposome Company, Inc. Methods of preparing low-toxicity drug-lipid complexes
US4775207A (en) 1987-03-17 1988-10-04 Bell Communications Research, Inc. Electro-optical switch
JP3101690B2 (ja) 1987-03-18 2000-10-23 エス・ビィ・２・インコーポレイテッド 変性抗体の、または変性抗体に関する改良
CA1331157C (fr) 1987-04-06 1994-08-02 Randal J. Kaufman Methode de production du facteur viii : proteines de type c
US6060447A (en) 1987-05-19 2000-05-09 Chiron Corporation Protein complexes having Factor VIII:C activity and production thereof
IE69026B1 (en) 1987-06-12 1996-08-07 Immuno Ag Novel proteins with factor VIII activity process for their preparation using genetically-engineered cells and pharmaceutical compositions containing them
US6346513B1 (en) 1987-06-12 2002-02-12 Baxter Trading Gmbh Proteins with factor VIII activity: process for their preparation using genetically-engineered cells and pharmaceutical compositions containing them
DE3720246A1 (de) 1987-06-19 1988-12-29 Behringwerke Ag Faktor viii:c-aehnliches molekuel mit koagulationsaktivitaet
US4897268A (en) 1987-08-03 1990-01-30 Southern Research Institute Drug delivery system and method of making the same
US4976696A (en) 1987-08-10 1990-12-11 Becton, Dickinson And Company Syringe pump and the like for delivering medication
FR2619314B1 (fr) 1987-08-11 1990-06-15 Transgene Sa Analogue du facteur viii, procede de preparation et composition pharmaceutique le contenant
US4861800A (en) 1987-08-18 1989-08-29 Buyske Donald A Method for administering the drug deprenyl so as to minimize the danger of side effects
US4994371A (en) 1987-08-28 1991-02-19 Davie Earl W DNA preparation of Christmas factor and use of DNA sequences
AU598958B2 (en) 1987-11-12 1990-07-05 Vestar, Inc. Improved amphotericin b liposome preparation
US5270176A (en) 1987-11-20 1993-12-14 Hoechst Aktiengesellschaft Method for the selective cleavage of fusion proteins with lysostaphin
GB8807504D0 (en) 1988-03-29 1988-05-05 Sandoz Ltd Improvements in/relating to organic compounds
US5037743A (en) 1988-08-05 1991-08-06 Zymogenetics, Inc. BAR1 secretion signal
JP2717808B2 (ja) 1988-08-10 1998-02-25 テルモ株式会社 シリンジポンプ
US5223409A (en) 1988-09-02 1993-06-29 Protein Engineering Corp. Directed evolution of novel binding proteins
US6780613B1 (en) 1988-10-28 2004-08-24 Genentech, Inc. Growth hormone variants
US5534617A (en) 1988-10-28 1996-07-09 Genentech, Inc. Human growth hormone variants having greater affinity for human growth hormone receptor at site 1
US5004803A (en) 1988-11-14 1991-04-02 Genetics Institute, Inc. Production of procoagulant proteins
CA2006596C (fr) 1988-12-22 2000-09-05 Rika Ishikawa G-csf modifie chimiquement
ATE148171T1 (de) 1989-02-21 1997-02-15 Univ Washington Modifizierte formen von fortpflanzungshormonen
US5017378A (en) 1989-05-01 1991-05-21 The University Of Virginia Alumni Patents Foundation Intraorgan injection of biologically active compounds contained in slow-release microcapsules or microspheres
WO1990013361A1 (fr) 1989-05-04 1990-11-15 Southern Research Institute Procede ameliore d'encapsulation et produits obtenus
US5599907A (en) 1989-05-10 1997-02-04 Somatogen, Inc. Production and use of multimeric hemoglobins
US5298022A (en) 1989-05-29 1994-03-29 Amplifon Spa Wearable artificial pancreas
FR2647677B1 (fr) 1989-05-31 1991-09-27 Roussel Uclaf Nouvelles micro-proteines, procede de preparation et application a titre de medicaments de ces nouvelles micro-proteines
US7413537B2 (en) 1989-09-01 2008-08-19 Dyax Corp. Directed evolution of disulfide-bonded micro-proteins
SE465222C5 (sv) 1989-12-15 1998-02-10 Pharmacia & Upjohn Ab Ett rekombinant, humant faktor VIII-derivat och förfarande för dess framställning
US5492534A (en) 1990-04-02 1996-02-20 Pharmetrix Corporation Controlled release portable pump
US5318540A (en) 1990-04-02 1994-06-07 Pharmetrix Corporation Controlled release infusion device
US6552170B1 (en) 1990-04-06 2003-04-22 Amgen Inc. PEGylation reagents and compounds formed therewith
US5176502A (en) 1990-04-25 1993-01-05 Becton, Dickinson And Company Syringe pump and the like for delivering medication
US6517859B1 (en) 1990-05-16 2003-02-11 Southern Research Institute Microcapsules for administration of neuroactive agents
US5215680A (en) 1990-07-10 1993-06-01 Cavitation-Control Technology, Inc. Method for the production of medical-grade lipid-coated microbubbles, paramagnetic labeling of such microbubbles and therapeutic uses of microbubbles
US5583107A (en) 1990-09-04 1996-12-10 Cor Therapeutics, Inc. Agents affecting thrombosis and hemostasis
AU662508B2 (en) 1990-12-13 1995-09-07 Pharmacia & Upjohn Company Fusion polypeptides
US5206161A (en) 1991-02-01 1993-04-27 Genentech, Inc. Human plasma carboxypeptidase B
US20060122376A1 (en) 1991-02-07 2006-06-08 Chiron Corporation Protein complexes having factor VIII:C activity and production thereof
US5833982A (en) 1991-02-28 1998-11-10 Zymogenetics, Inc. Modified factor VII
SG47099A1 (en) 1991-03-15 1998-03-20 Amgen Boulder Inc Pegylation of polypeptides
US5846951A (en) 1991-06-06 1998-12-08 The School Of Pharmacy, University Of London Pharmaceutical compositions
IE922437A1 (en) 1991-07-25 1993-01-27 Idec Pharma Corp Recombinant antibodies for human therapy
US5859204A (en) 1992-04-07 1999-01-12 Emory University Modified factor VIII
US6376463B1 (en) 1992-04-07 2002-04-23 Emory University Modified factor VIII
US5364771A (en) 1992-04-07 1994-11-15 Emory University Hybrid human/porcine factor VIII
CA2124690C (fr) 1992-10-02 2007-09-11 Thomas Osterberg Composition contenant une preparation du facteur de coagulation viii, methode de preparation et utilisation d'un surfactant comme stabilisateur
US5563045A (en) 1992-11-13 1996-10-08 Genetics Institute, Inc. Chimeric procoagulant proteins
US5573776A (en) 1992-12-02 1996-11-12 Alza Corporation Oral osmotic device with hydrogel driving member
ES2113094T3 (es) 1993-03-09 1998-04-16 Epic Therapeutics Inc Las microparticulas macromoleculares y metodos de obtencion.
US5981719A (en) 1993-03-09 1999-11-09 Epic Therapeutics, Inc. Macromolecular microparticles and methods of production and use
US5554730A (en) * 1993-03-09 1996-09-10 Middlesex Sciences, Inc. Method and kit for making a polysaccharide-protein conjugate
US6090925A (en) 1993-03-09 2000-07-18 Epic Therapeutics, Inc. Macromolecular microparticles and methods of production and use
SE504074C2 (sv) 1993-07-05 1996-11-04 Pharmacia Ab Proteinberedning för subkutan, intramuskulär eller intradermal administrering
US5576291A (en) 1993-09-13 1996-11-19 Baxter International Inc. Activated factor VIII as a therapeutic agent and method of treating factor VIII deficiency
US5643575A (en) 1993-10-27 1997-07-01 Enzon, Inc. Non-antigenic branched polymer conjugates
US20020042079A1 (en) 1994-02-01 2002-04-11 Sanford M. Simon Methods and agents for measuring and controlling multidrug resistance
US5660848A (en) 1994-11-02 1997-08-26 The Population Council, Center For Biomedical Research Subdermally implantable device
GB9422383D0 (en) 1994-11-05 1995-01-04 Wellcome Found Antibodies
SE503424C2 (sv) 1994-11-14 1996-06-10 Pharmacia Ab Process för rening av rekombinant koagulationsfaktor VIII
US6818439B1 (en) 1994-12-30 2004-11-16 Chiron Corporation Methods for administration of recombinant gene delivery vehicles for treatment of hemophilia and other disorders
US6485726B1 (en) 1995-01-17 2002-11-26 The Brigham And Women's Hospital, Inc. Receptor specific transepithelial transport of therapeutics
US6086875A (en) 1995-01-17 2000-07-11 The Brigham And Women's Hospital, Inc. Receptor specific transepithelial transport of immunogens
US6030613A (en) 1995-01-17 2000-02-29 The Brigham And Women's Hospital, Inc. Receptor specific transepithelial transport of therapeutics
US6096871A (en) 1995-04-14 2000-08-01 Genentech, Inc. Polypeptides altered to contain an epitope from the Fc region of an IgG molecule for increased half-life
US5869046A (en) 1995-04-14 1999-02-09 Genentech, Inc. Altered polypeptides with increased half-life
US5739277A (en) 1995-04-14 1998-04-14 Genentech Inc. Altered polypeptides with increased half-life
US6121022A (en) 1995-04-14 2000-09-19 Genentech, Inc. Altered polypeptides with increased half-life
US7846455B2 (en) 1996-07-15 2010-12-07 The United States Of America As Represented By The Department Of Health And Human Services Attenuated chimeric respiratory syncytial virus
SE9503380D0 (sv) 1995-09-29 1995-09-29 Pharmacia Ab Protein derivatives
GB9526733D0 (en) 1995-12-30 1996-02-28 Delta Biotechnology Ltd Fusion proteins
SI0932399T1 (sl) 1996-03-12 2006-10-31 Pg Txl Co Lp Vodotopna paklitakselna predzdravila
US6441025B2 (en) * 1996-03-12 2002-08-27 Pg-Txl Company, L.P. Water soluble paclitaxel derivatives
US8183344B2 (en) 1996-04-24 2012-05-22 University Of Michigan Inactivation resistant factor VIII
US7560107B2 (en) 1996-06-26 2009-07-14 Emory University Modified factor VIII
US6458563B1 (en) 1996-06-26 2002-10-01 Emory University Modified factor VIII
AU3968897A (en) 1996-08-02 1998-02-25 Bristol-Myers Squibb Company A method for inhibiting immunoglobulin-induced toxicity resulting from the use of immunoglobulins in therapy and in vivo diagnosis
DE69725747T2 (de) 1996-08-23 2004-07-29 Sequus Pharmaceuticals, Inc., Menlo Park Liposome enthaltend cisplatin
EP0932390A1 (fr) 1996-10-11 1999-08-04 Sequus Pharmaceuticals, Inc. Composition de liposomes therapeutiques et procede
US6056973A (en) 1996-10-11 2000-05-02 Sequus Pharmaceuticals, Inc. Therapeutic liposome composition and method of preparation
ES2321769T3 (es) 1996-10-15 2009-06-10 Transave, Inc. Liposomas de n-acil fosfatidiletanolamina para el transporte de medicamentos.
AU721653B2 (en) 1996-10-25 2000-07-13 Supernus Pharmaceuticals, Inc. Soluble form osmotic dose delivery system
US6361796B1 (en) 1996-10-25 2002-03-26 Shire Laboratories, Inc. Soluble form osmotic dose delivery system
WO1998022577A1 (fr) 1996-11-15 1998-05-28 Maria Grazia Masucci Proteines de fusion a demi-vie allongee
US6395302B1 (en) 1996-11-19 2002-05-28 Octoplus B.V. Method for the preparation of microspheres which contain colloidal systems
EP0842657A1 (fr) 1996-11-19 1998-05-20 OctoPlus B.V. Microsphères pour la libération contrÔlée et procédés pour la préparation de telles microsphères
WO1998023289A1 (fr) 1996-11-27 1998-06-04 The General Hospital Corporation Modulation de la fixation de l'igg au fcrn
US6294170B1 (en) 1997-08-08 2001-09-25 Amgen Inc. Composition and method for treating inflammatory diseases
AT405516B (de) 1997-02-27 1999-09-27 Immuno Ag Faktor x-analoge mit modifizierter proteasespaltstelle
US6277375B1 (en) 1997-03-03 2001-08-21 Board Of Regents, The University Of Texas System Immunoglobulin-like domains with increased half-lives
CA2225189C (fr) 1997-03-06 2010-05-25 Queen's University At Kingston Gene et proteine du facteur viii du chien et methodes d'utilisation
DE69833755T2 (de) 1997-05-21 2006-12-28 Biovation Ltd. Verfahren zur herstellung von nicht-immunogenen proteinen
US6310183B1 (en) 1997-09-10 2001-10-30 Novo Nordisk A/S Coagulation factor VIIa composition
US7786070B2 (en) * 1997-09-10 2010-08-31 Novo Nordisk Healthcare A/G Subcutaneous administration of coagulation factor VII
DE19747261A1 (de) 1997-10-25 1999-04-29 Bayer Ag Osmotisches Arzneimittelfreisetzungssystem
GB9722131D0 (en) 1997-10-20 1997-12-17 Medical Res Council Method
DE69838526T2 (de) 1998-02-05 2008-07-03 Biosense Webster, Inc., Diamond Bar Gerät zum Freisetzen eines Medikaments im Herzen
US20050260605A1 (en) 1998-02-11 2005-11-24 Maxygen, Inc. Targeting of genetic vaccine vectors
EP1054973A1 (fr) 1998-02-11 2000-11-29 Maxygen, Inc. Immunisation par bibliotheque d'antigenes
DK1068241T3 (da) 1998-04-02 2008-02-04 Genentech Inc Antistofvarianter og fragmenter deraf
US6528624B1 (en) 1998-04-02 2003-03-04 Genentech, Inc. Polypeptide variants
US6242195B1 (en) 1998-04-02 2001-06-05 Genentech, Inc. Methods for determining binding of an analyte to a receptor
US6194551B1 (en) 1998-04-02 2001-02-27 Genentech, Inc. Polypeptide variants
US6406632B1 (en) 1998-04-03 2002-06-18 Symyx Technologies, Inc. Rapid characterization of polymers
GB9809951D0 (en) 1998-05-08 1998-07-08 Univ Cambridge Tech Binding molecules
US7090976B2 (en) 1999-11-10 2006-08-15 Rigel Pharmaceuticals, Inc. Methods and compositions comprising Renilla GFP
GB9815157D0 (en) 1998-07-13 1998-09-09 Metron Designs Ltd High resolution pulse width setting from relatively low frequency clocks
JP2002522063A (ja) 1998-08-17 2002-07-23 アブジェニックス インコーポレイテッド 増加した血清半減期を有する改変された分子の生成
US20030190740A1 (en) 1998-10-13 2003-10-09 The University Of Georgia Research Foundation, Inc Stabilized bioactive peptides and methods of identification, synthesis, and use
DK1129186T4 (da) 1998-11-10 2017-02-06 Stichting Sanquin Bloedvoorziening Et faktor VIII-polypeptid med faktor VIII:C-aktivitet
EP1006183A1 (fr) 1998-12-03 2000-06-07 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Récepteurs Fc recombinantes et solubles
US6329186B1 (en) 1998-12-07 2001-12-11 Novozymes A/S Glucoamylases with N-terminal extensions
US6358703B1 (en) 1998-12-10 2002-03-19 Bayer Corporation Expression system for factor VIII
US6713086B2 (en) 1998-12-18 2004-03-30 Abbott Laboratories Controlled release formulation of divalproex sodium
KR101155191B1 (ko) 1999-01-15 2012-06-13 제넨테크, 인크. 효과기 기능이 변화된 폴리펩티드 변이체
US6737056B1 (en) 1999-01-15 2004-05-18 Genentech, Inc. Polypeptide variants with altered effector function
AU766410B2 (en) 1999-03-03 2003-10-16 Optinose As Nasal delivery device
US6183770B1 (en) 1999-04-15 2001-02-06 Acutek International Carrier patch for the delivery of agents to the skin
US7666400B2 (en) * 2005-04-06 2010-02-23 Ibc Pharmaceuticals, Inc. PEGylation by the dock and lock (DNL) technique
US6743211B1 (en) 1999-11-23 2004-06-01 Georgia Tech Research Corporation Devices and methods for enhanced microneedle penetration of biological barriers
US7829085B2 (en) 1999-07-14 2010-11-09 Life Sciences Research Partners Vzw Methods of treating hemostasis disorders using antibodies binding the C1 domain of factor VIII
CA2380633A1 (fr) 1999-08-06 2001-02-15 Genentech, Inc. Antagoniste du fviia
US6458387B1 (en) 1999-10-18 2002-10-01 Epic Therapeutics, Inc. Sustained release microspheres
US20030109428A1 (en) 1999-12-01 2003-06-12 John Bertin Novel molecules of the card-related protein family and uses thereof
US20050287153A1 (en) * 2002-06-28 2005-12-29 Genentech, Inc. Serum albumin binding peptides for tumor targeting
US6352721B1 (en) 2000-01-14 2002-03-05 Osmotica Corp. Combined diffusion/osmotic pumping drug delivery system
WO2001058957A2 (fr) 2000-02-11 2001-08-16 Lexigen Pharmaceuticals Corp. Amelioration de la demi-vie circulante de proteines de fusion a base d'anticorps
WO2001058935A2 (fr) 2000-02-11 2001-08-16 Maxygen Aps MOLECULES DE TYPE FACTEUR VII OU VIIa
CA2405557C (fr) * 2000-04-12 2013-09-24 Human Genome Sciences, Inc. Proteines hybrides d'albumine
US6905683B2 (en) 2000-05-03 2005-06-14 Novo Nordisk Healthcare A/G Human coagulation factor VII variants
ES2256234T3 (es) 2000-05-16 2006-07-16 Lipoxen Technologies Limited Derivatizacion de proteinas en solucion acuosa.
AU2001284982A1 (en) 2000-08-15 2002-02-25 Board Of Trustees Of The University Of Illinois Method of forming microparticles
US7176288B2 (en) 2000-09-13 2007-02-13 Novo Nordisk Healthcare A/G Human coagulation factor VII variants
EP1319016A4 (fr) 2000-09-19 2006-05-10 Univ Emory Facteur viii modifie
DE10053224A1 (de) 2000-10-26 2002-05-08 Univ Goettingen Georg August Verfahren zur Exposition von Peptiden und Polypeptiden auf der Zelloberfläche von Bakterien
US20030228309A1 (en) 2000-11-08 2003-12-11 Theodora Salcedo Antibodies that immunospecifically bind to TRAIL receptors
AU2002226028A1 (en) 2000-11-14 2002-05-27 Board Of Regents, Unversity Of Texas Systems Mutant human factor ix with an increased resistance to inhibition by heparin
GB0029407D0 (en) 2000-12-01 2001-01-17 Affitech As Product
DE60143544D1 (de) 2000-12-12 2011-01-05 Medimmune Llc Moleküle mit längeren halbwertszeiten, zusammensetzungen und deren verwendung
US20030049689A1 (en) * 2000-12-14 2003-03-13 Cynthia Edwards Multifunctional polypeptides
US20030104591A1 (en) 2000-12-14 2003-06-05 Murray Christopher J. Methods and compositions for grafting functional loops into a protein
EP1354042A2 (fr) * 2000-12-29 2003-10-22 Curagen Corporation Proteines et acides nucleiques codant celles-ci
IN190699B (fr) 2001-02-02 2003-08-16 Sun Pharmaceutical Ind Ltd
IL156842A0 (en) * 2001-02-05 2004-02-08 Novo Nordisk Healthcare Ag Combined use of factor vii polypeptides and factor ix polypeptides
US6888319B2 (en) 2001-03-01 2005-05-03 Palomar Medical Technologies, Inc. Flashlamp drive circuit
US7211395B2 (en) 2001-03-09 2007-05-01 Dyax Corp. Serum albumin binding moieties
PE20020908A1 (es) * 2001-03-21 2002-10-26 Cell Therapeutics Inc Produccion recombinante de polimeros polianionicos y uso de de los mismos
US6992174B2 (en) 2001-03-30 2006-01-31 Emd Lexigen Research Center Corp. Reducing the immunogenicity of fusion proteins
US20040106794A1 (en) 2001-04-16 2004-06-03 Schering Corporation 3,4-Di-substituted cyclobutene-1,2-diones as CXC-chemokine receptor ligands
US20050048512A1 (en) 2001-04-26 2005-03-03 Avidia Research Institute Combinatorial libraries of monomer domains
WO2002087621A1 (fr) 2001-04-30 2002-11-07 Shire Laboratories Inc. Preparation pharmaceutique comprenant des inhibiteurs de l'ace/nep et des renforcateurs de biodisponibilite
US6838093B2 (en) 2001-06-01 2005-01-04 Shire Laboratories, Inc. System for osmotic delivery of pharmaceutically active agents
CA2450125A1 (fr) 2001-06-15 2002-12-27 Andre C. Schuh Therapie genique de l'hemophilie a
KR100407467B1 (ko) 2001-07-12 2003-11-28 최수봉 리모컨 방식의 인슐린 자동주사기
AU2002322394A1 (en) * 2001-07-30 2003-02-17 Eli Lilly And Company Method for treating diabetes and obesity
CN101724075B (zh) 2001-10-10 2014-04-30 诺和诺德公司 肽的重构和糖缀合
US7125843B2 (en) * 2001-10-19 2006-10-24 Neose Technologies, Inc. Glycoconjugates including more than one peptide
US6960657B2 (en) 2001-11-02 2005-11-01 Novo Nordisk Healthcare A/G Human coagulation factor VII polypeptides
CN100463962C (zh) 2001-12-07 2009-02-25 图尔金株式会社 嵌合蛋白质的表型筛选
EP2261250B1 (fr) 2001-12-21 2015-07-01 Human Genome Sciences, Inc. Protéines de fusion d'albumine et GCSF
US20080194481A1 (en) 2001-12-21 2008-08-14 Human Genome Sciences, Inc. Albumin Fusion Proteins
US20080167238A1 (en) 2001-12-21 2008-07-10 Human Genome Sciences, Inc. Albumin Fusion Proteins
KR101271635B1 (ko) 2001-12-21 2013-06-12 휴먼 게놈 사이언시즈, 인코포레이티드 알부민 융합 단백질
US20040002587A1 (en) 2002-02-20 2004-01-01 Watkins Jeffry D. Fc region variants
US7317091B2 (en) 2002-03-01 2008-01-08 Xencor, Inc. Optimized Fc variants
US20040132101A1 (en) 2002-09-27 2004-07-08 Xencor Optimized Fc variants and methods for their generation
KR20040088572A (ko) 2002-03-01 2004-10-16 이뮤노메딕스, 인코오포레이티드 제거율 증강을 위한 양특이성 항체 점 돌연변이들
EP1487992A4 (fr) 2002-03-15 2007-10-31 Brigham & Womens Hospital Apport par les voies aeriennes centrales pour l'administration systemique de medicaments
DK1499719T3 (da) 2002-04-30 2011-02-28 Bayer Healthcare Llc Faktor VII- eller VIIa-polypeptidvarianter
US6945952B2 (en) 2002-06-25 2005-09-20 Theraject, Inc. Solid solution perforator for drug delivery and other applications
US7294513B2 (en) 2002-07-24 2007-11-13 Wyatt Technology Corporation Method and apparatus for characterizing solutions of small particles
US7425620B2 (en) 2002-08-14 2008-09-16 Scott Koenig FcγRIIB-specific antibodies and methods of use thereof
WO2004027901A2 (fr) 2002-09-17 2004-04-01 Diffusion Science, Inc. Generation, stockage et reaction electrochimiques d'hydrogene et d'oxygene
US6911323B2 (en) 2002-09-25 2005-06-28 Novo Nordisk Healthcare A/G Human coagulation factor VII polypeptides
EP2364996B1 (fr) 2002-09-27 2016-11-09 Xencor Inc. Variantes FC optimisées et leurs procédés de génération
SI1562972T1 (sl) 2002-10-15 2010-12-31 Facet Biotech Corp ALTERACIJA FcRn VEZANIH AFINITET ALI SERUMSKIH RAZPOLOVNIH DOB ANTITELESC Z MUTAGENEZO
GB2395337B (en) 2002-11-14 2005-12-28 Gary Michael Wilson Warning Unit
ES2897506T3 (es) 2003-01-09 2022-03-01 Macrogenics Inc Identificación y modificación de anticuerpos con regiones Fc variantes y métodos de utilización de los mismos
US7041635B2 (en) 2003-01-28 2006-05-09 In2Gen Co., Ltd. Factor VIII polypeptide
CN102139114A (zh) 2003-02-26 2011-08-03 尼克塔治疗公司 聚合物-因子viii部分缀合物
WO2004076522A1 (fr) 2003-02-28 2004-09-10 Kuraray Co., Ltd. Composition de resine durcissable
US20090010920A1 (en) 2003-03-03 2009-01-08 Xencor, Inc. Fc Variants Having Decreased Affinity for FcyRIIb
US8388955B2 (en) 2003-03-03 2013-03-05 Xencor, Inc. Fc variants
US20050176108A1 (en) * 2003-03-13 2005-08-11 Young-Min Kim Physiologically active polypeptide conjugate having prolonged in vivo half-life
US7348004B2 (en) 2003-05-06 2008-03-25 Syntonix Pharmaceuticals, Inc. Immunoglobulin chimeric monomer-dimer hybrids
TWI353991B (en) 2003-05-06 2011-12-11 Syntonix Pharmaceuticals Inc Immunoglobulin chimeric monomer-dimer hybrids
US7132398B2 (en) 2003-05-06 2006-11-07 Dendreon Corporation Method of treatment of hemorrhagic disease using a factor VIIa/tissue factor inhibitor
US20070161087A1 (en) 2003-05-29 2007-07-12 Wolfgang Glaesner Glp-1 fusion proteins
US7498024B2 (en) 2003-06-03 2009-03-03 Cell Genesys, Inc. Compositions and methods for enhanced expression of immunoglobulins from a single vector using a peptide cleavage site
EP1654290B1 (fr) 2003-08-12 2019-03-13 Lipoxen Technologies Limited Derive d'acide sialique destine a la derivatisation et a la conjugaison proteinique
JP4934796B2 (ja) 2003-08-14 2012-05-16 トロンボジェニクス・ナムローゼ・フエンノートシャップ 可変抗体
US20050152979A1 (en) 2003-09-05 2005-07-14 Cell Therapeutics, Inc. Hydrophobic drug compositions containing reconstitution enhancer
CN101870729A (zh) 2003-09-09 2010-10-27 诺和诺德医疗保健公司 凝固因子ⅶ多肽
BRPI0414539B8 (pt) 2003-09-19 2021-05-25 Novo Nordisk As composto, composição farmacêutica, e, uso de um composto
GB0324368D0 (en) 2003-10-17 2003-11-19 Univ Cambridge Tech Polypeptides including modified constant regions
EP1682106A4 (fr) 2003-10-30 2008-06-11 Univ Emory Facteur fviii modifie presentant une immunogenicite reduite par mutagenese d'epitopes a2 et c2
US7211559B2 (en) 2003-10-31 2007-05-01 University Of Maryland, Baltimore Factor VIII compositions and methods
WO2005047327A2 (fr) 2003-11-12 2005-05-26 Biogen Idec Ma Inc. Variants de polypeptide se liant au recepteur fc neonatal (fcrn), proteines de liaison fc dimeres et techniques associees
WO2005051289A2 (fr) * 2003-11-18 2005-06-09 Iconic Therapeutics, Inc. Preparations homogenes de proteines chimeres
US20060040856A1 (en) * 2003-12-03 2006-02-23 Neose Technologies, Inc. Glycopegylated factor IX
WO2005077981A2 (fr) 2003-12-22 2005-08-25 Xencor, Inc. Polypeptides fc a nouveaux sites de liaison de ligands fc
WO2005070963A1 (fr) 2004-01-12 2005-08-04 Applied Molecular Evolution, Inc Variants de la region fc
US20060026719A1 (en) 2004-01-14 2006-02-02 Kieliszewski Marcia J Methods of producing peptides/proteins in plants and peptides/proteins produced thereby
EP1729795B1 (fr) * 2004-02-09 2016-02-03 Human Genome Sciences, Inc. Proteines hybrides d'albumine
US8076288B2 (en) * 2004-02-11 2011-12-13 Amylin Pharmaceuticals, Inc. Hybrid polypeptides having glucose lowering activity
US7276585B2 (en) 2004-03-24 2007-10-02 Xencor, Inc. Immunoglobulin variants outside the Fc region
WO2005123780A2 (fr) 2004-04-09 2005-12-29 Protein Design Labs, Inc. Modification des affinites de liaison pour le fcrn ou de la demi-vie serique d'anticorps par mutagenese
US20080233100A1 (en) 2004-06-30 2008-09-25 Yiyou Chen Targeted enzymes
WO2006085967A2 (fr) 2004-07-09 2006-08-17 Xencor, Inc. Anticorps monoclonaux optimises anti-cd20 a variants fc
SI2471813T1 (sl) 2004-07-15 2015-03-31 Xencor, Inc. Optimirane Fc variante
US7566701B2 (en) 2004-09-07 2009-07-28 Archemix Corp. Aptamers to von Willebrand Factor and their use as thrombotic disease therapeutics
CA2580796C (fr) * 2004-09-24 2013-03-26 Amgen Inc. Molecules fc modifiees ayant des peptides inserees dans les regions de boucle interne
WO2006047350A2 (fr) 2004-10-21 2006-05-04 Xencor, Inc. Variants d'immunoglobuline igg a fonction effectrice optimisee
PT2371856T (pt) 2004-11-12 2022-08-12 Bayer Healthcare Llc Modificação de fviii direcionada a sítio
EP1835938B1 (fr) 2004-12-27 2013-08-07 Baxter International Inc. Conjugues entre polymeres et facteurs von willebrand
EP1841787A2 (fr) 2005-01-25 2007-10-10 Cell Therapeutics, Inc. Conjugues de proteine biologiquement actives possedant une demi-vie in vivo modifiee
US8263545B2 (en) 2005-02-11 2012-09-11 Amylin Pharmaceuticals, Inc. GIP analog and hybrid polypeptides with selectable properties
WO2006103298A2 (fr) 2005-04-01 2006-10-05 Novo Nordisk Health Care Ag Analogues du fviii de coagulation sanguine
JP2008537680A (ja) 2005-04-14 2008-09-25 ツェー・エス・エル・ベーリング・ゲー・エム・ベー・ハー 安定性の増大された改変型凝固第ｖｉｉｉ因子およびその誘導体
EP1924596A4 (fr) 2005-08-12 2009-07-29 Human Genome Sciences Inc Protéines de fusion avec l'albumine
US20090011992A1 (en) 2005-09-21 2009-01-08 Novo Nordisk Healthcare A/G Human Coagulation Factor VII Polypeptides
US20090099031A1 (en) * 2005-09-27 2009-04-16 Stemmer Willem P Genetic package and uses thereof
US7846445B2 (en) 2005-09-27 2010-12-07 Amunix Operating, Inc. Methods for production of unstructured recombinant polymers and uses thereof
JP2009509535A (ja) 2005-09-27 2009-03-12 アムニクス， インコーポレイテッド タンパク様薬剤およびその使用
US7855279B2 (en) 2005-09-27 2010-12-21 Amunix Operating, Inc. Unstructured recombinant polymers and uses thereof
US20130172274A1 (en) 2005-12-20 2013-07-04 Duke University Methods and compositions for delivering active agents with enhanced pharmacological properties
EP1971355B1 (fr) 2005-12-20 2020-03-11 Duke University Procedes et compositions pour l'administration d'agents actifs a proprietes pharmacologiques ameliorees
US8048848B2 (en) 2006-02-03 2011-11-01 Prolor Biotech Ltd. Long-acting interferons and derivatives thereof and methods thereof
EP1816201A1 (fr) * 2006-02-06 2007-08-08 CSL Behring GmbH Facteur de coagulation VIIa modifié ayant une stabilité 'half-life' améliorée
FR2897868B1 (fr) 2006-02-24 2012-08-31 Lab Francais Du Fractionnement Anticorps anti-idiotypiques neutralisant l'activite inhibitrice d'un anticorps inhibiteur dirige contre le domaine c1 du facteur viii.
CA2643182C (fr) 2006-03-13 2014-09-02 Tir Technology Lp Appareil et procede de commande adaptative pour systeme d'eclairage a semi-conducteurs
US7683158B2 (en) 2006-03-31 2010-03-23 Baxter International Inc. Pegylated factor VIII
US7645860B2 (en) 2006-03-31 2010-01-12 Baxter Healthcare S.A. Factor VIII polymer conjugates
WO2007130535A2 (fr) 2006-05-04 2007-11-15 Molecular Innovations Nouvelle technologie de fusion/étiquetage de protéines
WO2007144173A1 (fr) 2006-06-14 2007-12-21 Csl Behring Gmbh Protéine de fusion pouvant subir un clivage protéolytique et contenant un facteur de coagulation sanguine
EP1867660A1 (fr) 2006-06-14 2007-12-19 CSL Behring GmbH Protéine de fusion qui peut être clivée protéolyticalement et qui contient un facteur de la coagulation sanguine
US7939632B2 (en) 2006-06-14 2011-05-10 Csl Behring Gmbh Proteolytically cleavable fusion proteins with high molar specific activity
US7812121B2 (en) 2006-08-31 2010-10-12 Baker Audrey E GLP-2 mimetibodies, polypeptides, compositions, methods and uses
EP2068905A4 (fr) 2006-09-14 2009-12-30 Human Genome Sciences Inc Protéines de fusion d'albumine
US7985783B2 (en) 2006-09-21 2011-07-26 The Regents Of The University Of California Aldehyde tags, uses thereof in site-specific protein modification
WO2008049711A1 (fr) 2006-10-27 2008-05-02 Novo Nordisk A/S Insuline à peptide ajouté
CN101190945A (zh) 2006-11-29 2008-06-04 中国人民解放军军事医学科学院放射与辐射医学研究所 特异性抗凝血物质的制备及其应用
CA2673459C (fr) 2006-12-22 2016-09-13 Stefan Schulte Facteurs de coagulation modifies presentant une demi-vie in vivo prolongee
EP1935430A1 (fr) 2006-12-22 2008-06-25 CSL Behring GmbH Facteurs de coagulation modifiés avec une demi-vie in vivo prolongée
NZ577728A (en) * 2006-12-27 2012-01-12 Baxter Int Von willebrand factor- and factor viii-polymer conjugates having a releasable linkage
EP2099475B1 (fr) 2007-01-03 2016-08-24 Novo Nordisk Health Care AG Administration sous-cutanée de polypeptides liés au facteur viia de la coagulation
US7700734B2 (en) 2007-01-09 2010-04-20 Shu-Wha Lin Recombinant human factor IX and use thereof
EP2108045B1 (fr) 2007-02-01 2013-12-11 Baxter International Inc. Protéines mutantes de fix améliorées destinées à traiter l'hémophilie b
EP2517714A1 (fr) 2007-04-26 2012-10-31 Inspiration Biopharmaceuticals, Inc. Protéines dépendantes de la vitamine K recombinées à teneur élevée en acide sialique et leurs procédés de préparation
AU2008287340A1 (en) 2007-08-15 2009-02-19 Amunix, Inc. Compositions and methods for modifying properties of biologically active polypeptides
CA2703948A1 (fr) 2007-11-01 2009-05-07 University Of Rochester Facteur viii recombinant ayant une stabilite accrue
US8173597B2 (en) 2007-11-09 2012-05-08 Baxter International Inc. Modified recombinant factor VIII and von Willebrand factor and methods of use
EP3936116A1 (fr) 2007-12-28 2022-01-12 Takeda Pharmaceutical Company Limited Formulations comprenant de vwf recombinant
JP5444629B2 (ja) 2008-04-03 2014-03-19 富士通株式会社 照度センサ用光案内機構及び携帯電話機
AR071478A1 (es) 2008-04-17 2010-06-23 Baxter Healthcare Sa Peptidos de bajo peso molecular con actividad procoagulante para el tratamiento de pacientes con deficiencia de factor v (fv), fvii, fviii, fx y/o fxi
MX2010013219A (es) 2008-06-04 2011-04-11 Bayer Healthcare Llc Muteínas de fviii para el tratamiento de la enfermedad de von willebrand.
CA2728012C (fr) 2008-06-24 2017-10-31 Csl Behring Gmbh Facteur viii, facteur de von willebrand ou leurs complexes avec des durees de vies in vivo prolongees
CA2740793A1 (fr) 2008-11-03 2010-06-03 Haiyan Jiang Procede de traitement de l'hemophilie
EP2368124A4 (fr) 2008-11-24 2012-09-19 Bayer Healthcare Llc Procédé de détermination d'activité de facteur de coagulation sanguine pégylé dans un essai de temps de thromboplastine partielle activée à base de silice
US8680050B2 (en) 2009-02-03 2014-03-25 Amunix Operating Inc. Growth hormone polypeptides fused to extended recombinant polypeptides and methods of making and using same
US8703717B2 (en) 2009-02-03 2014-04-22 Amunix Operating Inc. Growth hormone polypeptides and methods of making and using same
CN102348715B (zh) 2009-02-03 2017-12-08 阿穆尼克斯运营公司 延伸重组多肽和包含该延伸重组多肽的组合物
US20120142593A1 (en) 2009-03-24 2012-06-07 Bayer Healthcare Llc Factor VIII Variants and Methods of Use
CN106916229A (zh) 2009-06-08 2017-07-04 阿穆尼克斯运营公司 生长激素多肽及其制备和使用方法
NZ596778A (en) 2009-06-08 2013-11-29 Amunix Operating Inc Glucose-regulating polypeptides and methods of making and using same
AU2010284977A1 (en) 2009-08-20 2012-03-29 Csl Behring Gmbh Albumin fused coagulation factors for non-intravenous administration in the therapy and prophylactic treatment of bleeding disorders
AU2010290077C1 (en) 2009-08-24 2015-12-03 Bioverativ Therapeutics Inc. Coagulation factor IX compositions and methods of making and using same
WO2011028344A2 (fr) 2009-08-25 2011-03-10 Amunix Operating Inc. Compositions d'antagonistes des récepteurs d'interleukine-1 et leurs procédés de préparation et d'utilisation
PL2499165T3 (pl) 2009-11-13 2017-04-28 Grifols Therapeutics Inc. Preparaty zawierające czynnik von willenbranda (vwf) oraz sposoby, zestawy i zastosowania z nim powiązane
AU2010325787B2 (en) 2009-12-06 2016-05-12 Bioverativ Therapeutics Inc. Factor VIII-Fc chimeric and hybrid polypeptides, and methods of use thereof
US20110312881A1 (en) * 2009-12-21 2011-12-22 Amunix, Inc. Bifunctional polypeptide compositions and methods for treatment of metabolic and cardiovascular diseases
EP2977055A1 (fr) 2010-02-16 2016-01-27 Novo Nordisk A/S Protéines de fusion de facteur viii
CN102770449B (zh) 2010-02-16 2016-02-24 诺沃—诺迪斯克有限公司 具有降低的vwf结合的因子viii分子
WO2011123830A2 (fr) 2010-04-02 2011-10-06 Amunix Operating Inc. Compositions d'alpha 1-antitrypsine, leurs procédés de préparation et d'utilisation
EP3372617B1 (fr) 2010-04-02 2024-07-24 Amunix Pharmaceuticals, Inc. Protéines de fusion liantes, conjugués protéines de fusion liantes-médicaments, conjugués xten-médicaments et procédés pour les préparer et les utiliser
EP2730663B1 (fr) 2010-04-21 2023-03-22 Erik Berntorp Facteurs génétiques associés au développement d'inhibiteur dans l'hémophilie A
NZ605400A (en) 2010-07-09 2015-05-29 Biogen Idec Hemophilia Inc Chimeric clotting factors
EP3508573A1 (fr) 2010-07-09 2019-07-10 Bioverativ Therapeutics Inc. Systèmes de traitement de facteur viii et procédés associés
NZ605348A (en) 2010-07-09 2015-01-30 Biogen Idec Hemophilia Inc Factor ix polypeptides and methods of use thereof
US20130183280A1 (en) 2010-07-15 2013-07-18 Novo Nordisk A/S Stabilized factor viii variants
US20130017997A1 (en) * 2010-08-19 2013-01-17 Amunix Operating Inc. Factor VIII Compositions and Methods of Making and Using Same
SI2717898T1 (sl) 2011-06-10 2019-07-31 Bioverativ Therapeutics Inc. Spojine prokoagulantov in metode za njihovo uporabo
WO2013106787A1 (fr) 2012-01-12 2013-07-18 Biogen Idec Ma Inc. Facteur viii chimérique et polypeptides et leurs utilisations
NZ628014A (en) 2012-02-15 2016-09-30 Biogen Ma Inc Recombinant factor viii proteins
RS63870B1 (sr) 2012-02-15 2023-01-31 Bioverativ Therapeutics Inc Sastavi faktora viii i postupci za pravljenje i upotrebu istih
CN104411323A (zh) 2012-04-24 2015-03-11 诺和诺德A/S（股份有限公司） 适用于治疗血友病的药物组合物
JP2015515482A (ja) 2012-04-24 2015-05-28 ノヴォ ノルディスク アー／エス 血友病の治療に適する化合物
KR102403545B1 (ko) 2012-07-11 2022-05-30 바이오버라티브 테라퓨틱스 인크. Xten 및 폰 빌레브란트 인자 단백질과의 viii 인자 복합체 및 이의 용도
CN104619677B (zh) 2012-07-18 2016-10-26 西北大学 形成乙烯的方法
WO2014100913A1 (fr) 2012-12-24 2014-07-03 Beijing Anxinhuaide Biotech. Co., Ltd Amélioration de la demi-vie d'un polypeptide thérapeutique par fusion avec une protéine d'échafaudage trimère via un espaceur
ES2657291T3 (es) 2013-04-22 2018-03-02 Csl Ltd. Un complejo covalente de factor de von Willebrand y factor VIII asociado por un puente disulfuro
TW201536811A (zh) 2013-05-31 2015-10-01 Biogen Idec Inc 嵌合ｆｖｉｉ－ｘｔｅｎ分子及其用途
EP3008084A2 (fr) 2013-06-12 2016-04-20 Novo Nordisk A/S Composés convenant pour le traitement de l'hémophilie
SG10201913738YA (en) 2013-06-28 2020-03-30 Bioverativ Therapeutics Inc Thrombin cleavable linker with xten and its uses thereof
EP3033097B1 (fr) 2013-08-14 2021-03-10 Bioverativ Therapeutics Inc. Fusions de factor viii-xten et leurs utilisations.
WO2015070014A1 (fr) * 2013-11-08 2015-05-14 Biogen Idec Ma Inc. Composé de fusion procoagulant
CN117106095A (zh) 2014-01-10 2023-11-24 比奥贝拉蒂治疗公司 因子viii嵌合蛋白及其用途
WO2016004113A1 (fr) * 2014-06-30 2016-01-07 Biogen Ma Inc. Gène du facteur ix optimisé
TWI741992B (zh) * 2015-08-03 2021-10-11 美商百歐維拉提夫治療公司 因子ｉｘ融合蛋白以及其製備及使用方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007103515A2 (fr) * 2006-03-06 2007-09-13 Amunix, Inc. Polymères recombinés non structurés et utilisations correspondantes
WO2008155134A1 (fr) * 2007-06-21 2008-12-24 Technische Universität München Protéines biologiquement actives présentant une stabilité in vivo et/ou in vitro accrue

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
MASSIMO MORFINI: "secondary prophylaxis with factor IX concentrates: continuous infusion", BLOOD TRANSFUS, vol. 6, no. suppl 2, 28 February 2008 (2008-02-28), pages s21-s25, XP002699004, *
PAULA ALVAREZ ET AL: "Improving protein pharmacokinetics by genetic fusion to simple amino acid sequences", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, US, vol. 279, January 2004 (2004-01), pages 3375-3380, XP008131025, ISSN: 0021-9258, DOI: 10.1074/JBC.M311356200 *
SCHLAPSCHY MARTIN ET AL: "Fusion of a recombinant antibody fragment with a homo-amino-acid polymer: effects on biophysical properties and prolonged plasma half-life", PROTEIN ENGINEERING, DESIGN AND SELECTION, OXFORD JOURNAL, LONDON, GB , vol. 20, no. 6 26 June 2007 (2007-06-26), pages 273-284, XP002498446, ISSN: 1741-0126, DOI: 10.1093/PROTEIN/GZM020 Retrieved from the Internet: URL:10.1093/protein/gzm020 [retrieved on 2007-06-26] *
See also references of WO2011028229A1 *
SHEFFIELD W P ET AL: "Effects of genetic fusion of factor IX to albumin on in vivo clearance in mice and rabbits", BRITISH JOURNAL OF HAEMATOLOGY, WILEY-BLACKWELL PUBLISHING LTD, GB, vol. 126, no. 4, 1 August 2004 (2004-08-01), pages 565-573, XP002395198, ISSN: 0007-1048, DOI: 10.1111/J.1365-2141.2004.05106.X *

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US20210277378A1 (en) 2021-09-09
CN102741275B (zh) 2016-08-03
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BR112012004104A2 (pt) 2016-03-15
US20120263703A1 (en) 2012-10-18
JP2015052015A (ja) 2015-03-19
US20150344862A1 (en) 2015-12-03
AU2010290077A1 (en) 2012-03-22
EP2470670A4 (fr) 2013-07-10
US20170247676A1 (en) 2017-08-31
CN102741422B (zh) 2016-06-08
JP2013502459A (ja) 2013-01-24
JP5813641B2 (ja) 2015-11-17
US20140186327A1 (en) 2014-07-03
EP3222287A1 (fr) 2017-09-27
CN102741422A (zh) 2012-10-17
CA2772051C (fr) 2020-08-18
BR112012004104B1 (pt) 2022-01-04
AU2010290077B2 (en) 2015-07-30
US20140328819A1 (en) 2014-11-06
BR112012004094A2 (pt) 2016-03-08
US9062299B2 (en) 2015-06-23
AU2010290131A1 (en) 2012-03-22
JP2013502458A (ja) 2013-01-24
US9758776B2 (en) 2017-09-12
WO2011028228A1 (fr) 2011-03-10
AU2010290131C1 (en) 2015-12-03
EP2470670A1 (fr) 2012-07-04
EP2470559B1 (fr) 2017-03-22
CN102741275A (zh) 2012-10-17
US20120263701A1 (en) 2012-10-18
US20110046061A1 (en) 2011-02-24
US20160362672A1 (en) 2016-12-15
US9376672B2 (en) 2016-06-28
CA2772051A1 (fr) 2011-03-10
AU2010290131B2 (en) 2015-08-06
US20110046060A1 (en) 2011-02-24
US8716448B2 (en) 2014-05-06
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EP2470559A4 (fr) 2013-07-31

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